Sample records for protein sequence coverage
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De novo protein sequencing by combining top-down and bottom-up tandem mass spectra.
Liu, Xiaowen; Dekker, Lennard J M; Wu, Si; Vanduijn, Martijn M; Luider, Theo M; Tolić, Nikola; Kou, Qiang; Dvorkin, Mikhail; Alexandrova, Sonya; Vyatkina, Kira; Paša-Tolić, Ljiljana; Pevzner, Pavel A
2014-07-03
There are two approaches for de novo protein sequencing: Edman degradation and mass spectrometry (MS). Existing MS-based methods characterize a novel protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the protein. Because each tandem mass spectrum covers only a short peptide of the target protein, the key to high coverage protein sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resolution mass spectrometers have become accessible to many laboratories. These mass spectrometers are capable of analyzing molecules of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a protein. We propose an algorithm, TBNovo, for de novo protein sequencing by combining top-down and bottom-up MS. In TBNovo, a top-down tandem mass spectrum is utilized as a scaffold, and bottom-up tandem mass spectra are aligned to the scaffold to increase sequence coverage. Experiments on data sets of two proteins showed that TBNovo achieved high sequence coverage and high sequence accuracy.
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Confetti: A Multiprotease Map of the HeLa Proteome for Comprehensive Proteomics*
Guo, Xiaofeng; Trudgian, David C.; Lemoff, Andrew; Yadavalli, Sivaramakrishna; Mirzaei, Hamid
2014-01-01
Bottom-up proteomics largely relies on tryptic peptides for protein identification and quantification. Tryptic digestion often provides limited coverage of protein sequence because of issues such as peptide length, ionization efficiency, and post-translational modification colocalization. Unfortunately, a region of interest in a protein, for example, because of proximity to an active site or the presence of important post-translational modifications, may not be covered by tryptic peptides. Detection limits, quantification accuracy, and isoform differentiation can also be improved with greater sequence coverage. Selected reaction monitoring (SRM) would also greatly benefit from being able to identify additional targetable sequences. In an attempt to improve protein sequence coverage and to target regions of proteins that do not generate useful tryptic peptides, we deployed a multiprotease strategy on the HeLa proteome. First, we used seven commercially available enzymes in single, double, and triple enzyme combinations. A total of 48 digests were performed. 5223 proteins were detected by analyzing the unfractionated cell lysate digest directly; with 42% mean sequence coverage. Additional strong-anion exchange fractionation of the most complementary digests permitted identification of over 3000 more proteins, with improved mean sequence coverage. We then constructed a web application (https://proteomics.swmed.edu/confetti) that allows the community to examine a target protein or protein isoform in order to discover the enzyme or combination of enzymes that would yield peptides spanning a certain region of interest in the sequence. Finally, we examined the use of nontryptic digests for SRM. From our strong-anion exchange fractionation data, we were able to identify three or more proteotypic SRM candidates within a single digest for 6056 genes. Surprisingly, in 25% of these cases the digest producing the most observable proteotypic peptides was neither trypsin nor Lys-C. SRM analysis of Asp-N versus tryptic peptides for eight proteins determined that Asp-N yielded higher signal in five of eight cases. PMID:24696503
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Khafizov, Kamil; Madrid-Aliste, Carlos; Almo, Steven C; Fiser, Andras
2014-03-11
The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins--including proteins for which reliable homology models can be generated--on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long.
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Khafizov, Kamil; Madrid-Aliste, Carlos; Almo, Steven C.; Fiser, Andras
2014-01-01
The exponential growth of protein sequence data provides an ever-expanding body of unannotated and misannotated proteins. The National Institutes of Health-supported Protein Structure Initiative and related worldwide structural genomics efforts facilitate functional annotation of proteins through structural characterization. Recently there have been profound changes in the taxonomic composition of sequence databases, which are effectively redefining the scope and contribution of these large-scale structure-based efforts. The faster-growing bacterial genomic entries have overtaken the eukaryotic entries over the last 5 y, but also have become more redundant. Despite the enormous increase in the number of sequences, the overall structural coverage of proteins—including proteins for which reliable homology models can be generated—on the residue level has increased from 30% to 40% over the last 10 y. Structural genomics efforts contributed ∼50% of this new structural coverage, despite determining only ∼10% of all new structures. Based on current trends, it is expected that ∼55% structural coverage (the level required for significant functional insight) will be achieved within 15 y, whereas without structural genomics efforts, realizing this goal will take approximately twice as long. PMID:24567391
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NASA Astrophysics Data System (ADS)
Weisbrod, Chad R.; Kaiser, Nathan K.; Syka, John E. P.; Early, Lee; Mullen, Christopher; Dunyach, Jean-Jacques; English, A. Michelle; Anderson, Lissa C.; Blakney, Greg T.; Shabanowitz, Jeffrey; Hendrickson, Christopher L.; Marshall, Alan G.; Hunt, Donald F.
2017-09-01
High resolution mass spectrometry is a key technology for in-depth protein characterization. High-field Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables high-level interrogation of intact proteins in the most detail to date. However, an appropriate complement of fragmentation technologies must be paired with FTMS to provide comprehensive sequence coverage, as well as characterization of sequence variants, and post-translational modifications. Here we describe the integration of front-end electron transfer dissociation (FETD) with a custom-built 21 tesla FT-ICR mass spectrometer, which yields unprecedented sequence coverage for proteins ranging from 2.8 to 29 kDa, without the need for extensive spectral averaging (e.g., 60% sequence coverage for apo-myoglobin with four averaged acquisitions). The system is equipped with a multipole storage device separate from the ETD reaction device, which allows accumulation of multiple ETD fragment ion fills. Consequently, an optimally large product ion population is accumulated prior to transfer to the ICR cell for mass analysis, which improves mass spectral signal-to-noise ratio, dynamic range, and scan rate. We find a linear relationship between protein molecular weight and minimum number of ETD reaction fills to achieve optimum sequence coverage, thereby enabling more efficient use of instrument data acquisition time. Finally, real-time scaling of the number of ETD reactions fills during method-based acquisition is shown, and the implications for LC-MS/MS top-down analysis are discussed. [Figure not available: see fulltext.
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GFam: a platform for automatic annotation of gene families.
Sasidharan, Rajkumar; Nepusz, Tamás; Swarbreck, David; Huala, Eva; Paccanaro, Alberto
2012-10-01
We have developed GFam, a platform for automatic annotation of gene/protein families. GFam provides a framework for genome initiatives and model organism resources to build domain-based families, derive meaningful functional labels and offers a seamless approach to propagate functional annotation across periodic genome updates. GFam is a hybrid approach that uses a greedy algorithm to chain component domains from InterPro annotation provided by its 12 member resources followed by a sequence-based connected component analysis of un-annotated sequence regions to derive consensus domain architecture for each sequence and subsequently generate families based on common architectures. Our integrated approach increases sequence coverage by 7.2 percentage points and residue coverage by 14.6 percentage points higher than the coverage relative to the best single-constituent database within InterPro for the proteome of Arabidopsis. The true power of GFam lies in maximizing annotation provided by the different InterPro data sources that offer resource-specific coverage for different regions of a sequence. GFam's capability to capture higher sequence and residue coverage can be useful for genome annotation, comparative genomics and functional studies. GFam is a general-purpose software and can be used for any collection of protein sequences. The software is open source and can be obtained from http://www.paccanarolab.org/software/gfam/.
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NASA Astrophysics Data System (ADS)
Long, Ying; Wood, Troy D.
2015-01-01
Most enzymatic microreactors for protein digestion are based on trypsin, but proteins with hydrophobic segments may be difficult to digest because of the paucity of Arg and Lys residues. Microreactors based on pepsin, which is less specific than trypsin, can overcome this challenge. Here, an integrated immobilized pepsin microreactor (IPMR)/nanoelectrospray emitter is examined for its potential for peptide mapping. For myoglobin, equivalent sequence coverage is obtained in a thousandth the time of solution digestion with better sequence coverage. While sequence coverage of cytochrome c is lesser than solution in this short duration, more highly-charged peptic peptides are produced and a number of peaks are unidentified at low-resolution, suggesting that high-resolution mass spectrometry is needed to take full advantage of integrated IPMR/nanoelectrospray devices.
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Draft versus finished sequence data for DNA and protein diagnostic signature development
Gardner, Shea N.; Lam, Marisa W.; Smith, Jason R.; Torres, Clinton L.; Slezak, Tom R.
2005-01-01
Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10−3–10−5 (∼8× coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of ∼1% (3× to 6× coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures. PMID:16243783
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Expanding proteome coverage with orthogonal-specificity α-Lytic proteases
DOE Office of Scientific and Technical Information (OSTI.GOV)
Meyer, Jesse G.; Kim, Sangtae; Maltby, David A.
2014-03-01
Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains. Therefore, additional proteases that improve proteome coverage by cleavage at sequences complimentary to trypsin may increase proteome coverage. We demonstrate the novel application of two proteases for bottom-up proteomics: wild type alpha-lytic protease (WaLP), and an active site mutant of WaLP, M190A alpha-lytic protease (MaLP). We assess several relevant factors including MS/MS fragmentation, peptide length, peptide yield, and protease specificity. Bymore » combining data from separate digestions with trypsin, LysC, WaLP, and MaLP, proteome coverage was increased 101% compared to trypsin digestion alone. To demonstrate how the gained sequence coverage can access additional PTM information, we show identification of a number of novel phosphorylation sites in the S. pombe proteome and include an illustrative example from the protein MPD2, wherein two novel sites are identified, one in a tryptic peptide too short to identify and the other in a sequence devoid of tryptic sites. The specificity of WaLP and MaLP for aliphatic amino acid side chains was particularly valuable for coverage of membrane protein sequences, which increased 350% when the data from trypsin, LysC, WaLP, and MaLP were combined.« less
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Dynamics of domain coverage of the protein sequence universe.
Rekapalli, Bhanu; Wuichet, Kristin; Peterson, Gregory D; Zhulin, Igor B
2012-11-16
The currently known protein sequence space consists of millions of sequences in public databases and is rapidly expanding. Assigning sequences to families leads to a better understanding of protein function and the nature of the protein universe. However, a large portion of the current protein space remains unassigned and is referred to as its "dark matter". Here we suggest that true size of "dark matter" is much larger than stated by current definitions. We propose an approach to reducing the size of "dark matter" by identifying and subtracting regions in protein sequences that are not likely to contain any domain. Recent improvements in computational domain modeling result in a decrease, albeit slowly, in the relative size of "dark matter"; however, its absolute size increases substantially with the growth of sequence data.
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Dynamics of domain coverage of the protein sequence universe
2012-01-01
Background The currently known protein sequence space consists of millions of sequences in public databases and is rapidly expanding. Assigning sequences to families leads to a better understanding of protein function and the nature of the protein universe. However, a large portion of the current protein space remains unassigned and is referred to as its “dark matter”. Results Here we suggest that true size of “dark matter” is much larger than stated by current definitions. We propose an approach to reducing the size of “dark matter” by identifying and subtracting regions in protein sequences that are not likely to contain any domain. Conclusions Recent improvements in computational domain modeling result in a decrease, albeit slowly, in the relative size of “dark matter”; however, its absolute size increases substantially with the growth of sequence data. PMID:23157439
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Protein family clustering for structural genomics.
Yan, Yongpan; Moult, John
2005-10-28
A major goal of structural genomics is the provision of a structural template for a large fraction of protein domains. The magnitude of this task depends on the number and nature of protein sequence families. With a large number of bacterial genomes now fully sequenced, it is possible to obtain improved estimates of the number and diversity of families in that kingdom. We have used an automated clustering procedure to group all sequences in a set of genomes into protein families. Bench-marking shows the clustering method is sensitive at detecting remote family members, and has a low level of false positives. This comprehensive protein family set has been used to address the following questions. (1) What is the structure coverage for currently known families? (2) How will the number of known apparent families grow as more genomes are sequenced? (3) What is a practical strategy for maximizing structure coverage in future? Our study indicates that approximately 20% of known families with three or more members currently have a representative structure. The study indicates also that the number of apparent protein families will be considerably larger than previously thought: We estimate that, by the criteria of this work, there will be about 250,000 protein families when 1000 microbial genomes have been sequenced. However, the vast majority of these families will be small, and it will be possible to obtain structural templates for 70-80% of protein domains with an achievable number of representative structures, by systematically sampling the larger families.
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NASA Astrophysics Data System (ADS)
Nicolardi, Simone; Giera, Martin; Kooijman, Pieter; Kraj, Agnieszka; Chervet, Jean-Pierre; Deelder, André M.; van der Burgt, Yuri E. M.
2013-12-01
Particularly in the field of middle- and top-down peptide and protein analysis, disulfide bridges can severely hinder fragmentation and thus impede sequence analysis (coverage). Here we present an on-line/electrochemistry/ESI-FTICR-MS approach, which was applied to the analysis of the primary structure of oxytocin, containing one disulfide bridge, and of hepcidin, containing four disulfide bridges. The presented workflow provided up to 80 % (on-line) conversion of disulfide bonds in both peptides. With minimal sample preparation, such reduction resulted in a higher number of peptide backbone cleavages upon CID or ETD fragmentation, and thus yielded improved sequence coverage. The cycle times, including electrode recovery, were rapid and, therefore, might very well be coupled with liquid chromatography for protein or peptide separation, which has great potential for high-throughput analysis.
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Alternative Enzymes Lead to Improvements in Sequence Coverage and PTM Analysis
Hooper, Kyle; Rosenblatt, Michael; Urh, Marjeta; Saveliev, Sergei; Hosfield, Chris; Kobs, Gary; Ford, Michael; Jones, Richard; Amunugama, Ravi; Allen, David; Brazas, Robert
2013-01-01
The profiling of proteins using biological mass spectrometry (bottom up proteomics) most commonly requires trypsin. Trypsin is advantageous in that it produces peptides of optimal charge and size. However, for applications in which the proteins under investigation are part of a complex mixture or not isolated at high levels (i.e. low ng from an immunoprecipitation), sequence coverage is rarely complete. In addition, we have found that in several cases, like phosphorylation, acetylation, and methylation, alternative proteases are required to prepare peptides suitable for MS detection. This poster will provide specific examples which demonstrate this observation. For example, the application of a combined Trypsin/ Lys-C mixture reduces the number of missed cleavages by more than 3-fold producing samples with lower CV's (for biological replicates). The mixture is also well-suited for the complete proteolysis of hydrophobic, compact proteins. The addition of chymotrypsin and elastase has been found to be useful for identifying phosphorylation sites on proteins, especially on sequences where the site of phosphorylation inhibits trypsin (i.e. proximal to K or R). Many epigenetic applications have focused on histone modifications, like lysine acetylation and arginine methylation. Alternative proteases like Asp-N, Glu-C, and chymotrypsin have been especially useful given the fact that the modified K and R residues are resistant to c-terminal cleavage by trypsin. Finally, in the case of serum profiling, the addition of the endoglycosidase, PNGase F has been found to improve sequence coverage due to the removal of N-linked glycans.
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A limited universe of membrane protein families and folds
Oberai, Amit; Ihm, Yungok; Kim, Sanguk; Bowie, James U.
2006-01-01
One of the goals of structural genomics is to obtain a structural representative of almost every fold in nature. A recent estimate suggests that 70%–80% of soluble protein domains identified in the first 1000 genome sequences should be covered by about 25,000 structures—a reasonably achievable goal. As no current estimates exist for the number of membrane protein families, however, it is not possible to know whether family coverage is a realistic goal for membrane proteins. Here we find that virtually all polytopic helical membrane protein families are present in the already known sequences so we can make an estimate of the total number of families. We find that only ∼700 polytopic membrane protein families account for 80% of structured residues and ∼1700 cover 90% of structured residues. While apparently a finite and reachable goal, we estimate that it will likely take more than three decades to obtain the structures needed for 90% residue coverage, if current trends continue. PMID:16815920
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Data mining of enzymes using specific peptides
2009-01-01
Background Predicting the function of a protein from its sequence is a long-standing challenge of bioinformatic research, typically addressed using either sequence-similarity or sequence-motifs. We employ the novel motif method that consists of Specific Peptides (SPs) that are unique to specific branches of the Enzyme Commission (EC) functional classification. We devise the Data Mining of Enzymes (DME) methodology that allows for searching SPs on arbitrary proteins, determining from its sequence whether a protein is an enzyme and what the enzyme's EC classification is. Results We extract novel SP sets from Swiss-Prot enzyme data. Using a training set of July 2006, and test sets of July 2008, we find that the predictive power of SPs, both for true-positives (enzymes) and true-negatives (non-enzymes), depends on the coverage length of all SP matches (the number of amino-acids matched on the protein sequence). DME is quite different from BLAST. Comparing the two on an enzyme test set of July 2008, we find that DME has lower recall. On the other hand, DME can provide predictions for proteins regarded by BLAST as having low homologies with known enzymes, thus supplying complementary information. We test our method on a set of proteins belonging to 10 bacteria, dated July 2008, establishing the usefulness of the coverage-length cutoff to determine true-negatives. Moreover, sifting through our predictions we find that some of them have been substantiated by Swiss-Prot annotations by July 2009. Finally we extract, for production purposes, a novel SP set trained on all Swiss-Prot enzymes as of July 2009. This new set increases considerably the recall of DME. The new SP set is being applied to three metagenomes: Sargasso Sea with over 1,000,000 proteins, producing predictions of over 220,000 enzymes, and two human gut metagenomes. The outcome of these analyses can be characterized by the enzymatic profile of the metagenomes, describing the relative numbers of enzymes observed for different EC categories. Conclusions Employing SPs for predicting enzymatic activity of proteins works well once one utilizes coverage-length criteria. In our analysis, L ≥ 7 has led to highly accurate results. PMID:20034383
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Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin.
Floris, Federico; Chiron, Lionel; Lynch, Alice M; Barrow, Mark P; Delsuc, Marc-André; O'Connor, Peter B
2018-06-04
Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about ~23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from ~23% to ~42%. Graphical Abstract Two-dimensional mass spectrometry, when applied to primary fragment ions from the source, allows deep-sequencing of the protein calmodulin.
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Ruan, Guihua; Wei, Meiping; Chen, Zhengyi; Su, Rihui; Du, Fuyou; Zheng, Yanjie
2014-09-15
A novel large-volume immobilized enzyme reactor (IMER) on small column was prepared with organic-inorganic hybrid silica particles and applied for fast (10 min) and oriented digestion of protein. At first, a thin enzyme support layer was formed in the bottom of the small column by polymerization with α-methacrylic acid and dimethacrylate. After that, amino SiO2 particles was prepared by the sol-gel method with tetraethoxysilane and 3-aminopropyltriethoxysilane. Subsequently, the amino SiO2 particles were activated by glutaraldehyde for covalent immobilization of trypsin. Digestive capability of large-volume IMER for proteins was investigated by using bovine serum albumin (BSA), cytochrome c (Cyt-c) as model proteins. Results showed that although the sequence coverage of the BSA (20%) and Cyt-c (19%) was low, the large-volume IMER could produce peptides with stable specific sequence at 101-105, 156-160, 205-209, 212-218, 229-232, 257-263 and 473-451 of the amino sequence of BSA when digesting 1mg/mL BSA. Eight of common peptides were observed during each of the ten runs of large-volume IMER. Besides, the IMER could be easily regenerated by reactivating with GA and cross-linking with trypsin after breaking the -C=N- bond by 0.01 M HCl. The sequence coverage of BSA from regenerated IMER increased to 25% comparing the non-regenerated IMER (17%). 14 common peptides. accounting for 87.5% of first use of IMER, were produced both with IMER and regenerated IMER. When the IMER was applied for ginkgo albumin digestion, the sequence coverage of two main proteins of ginkgo, ginnacin and legumin, was 56% and 55%, respectively. (Reviewer 2) Above all, the fast and selective digestion property of the large-volume IMER indicated that the regenerative IMER could be tentatively used for the production of potential bioactive peptides and the study of oriented protein digestion. Copyright © 2014 Elsevier B.V. All rights reserved.
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Protein structure determination by exhaustive search of Protein Data Bank derived databases.
Stokes-Rees, Ian; Sliz, Piotr
2010-12-14
Parallel sequence and structure alignment tools have become ubiquitous and invaluable at all levels in the study of biological systems. We demonstrate the application and utility of this same parallel search paradigm to the process of protein structure determination, benefitting from the large and growing corpus of known structures. Such searches were previously computationally intractable. Through the method of Wide Search Molecular Replacement, developed here, they can be completed in a few hours with the aide of national-scale federated cyberinfrastructure. By dramatically expanding the range of models considered for structure determination, we show that small (less than 12% structural coverage) and low sequence identity (less than 20% identity) template structures can be identified through multidimensional template scoring metrics and used for structure determination. Many new macromolecular complexes can benefit significantly from such a technique due to the lack of known homologous protein folds or sequences. We demonstrate the effectiveness of the method by determining the structure of a full-length p97 homologue from Trichoplusia ni. Example cases with the MHC/T-cell receptor complex and the EmoB protein provide systematic estimates of minimum sequence identity, structure coverage, and structural similarity required for this method to succeed. We describe how this structure-search approach and other novel computationally intensive workflows are made tractable through integration with the US national computational cyberinfrastructure, allowing, for example, rapid processing of the entire Structural Classification of Proteins protein fragment database.
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Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing.
Zhang, Jin; Ruhlman, Tracey A; Mower, Jeffrey P; Jansen, Robert K
2013-12-29
Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling RNA from four tissue types rather than from a single tissue. Finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants.
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Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing
2013-01-01
Background Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Results Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. Conclusions The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling RNA from four tissue types rather than from a single tissue. Finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants. PMID:24373163
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Candidate mosaic proteins for a pan-filoviral cytotoxic T-Cell lymphocyte vaccine
DOE Office of Scientific and Technical Information (OSTI.GOV)
Fenimore, Paul W; Fischer, William M; Kuiken, Carla
The extremely high fatality rates of many filovirus (FILV) strains the recurrent but rarely identified origin of human epidemics, the only partly identified viral reservoirs and the continuing non-human primate epizootics in Africa make a broadly-protective filovirus vaccine highly desirable. Cytotoxic T-cells (CTL) have been shown to be protective in mice, guinea pigs and non-human primates. In murine models the cytotoxic T-cell epitopes that are protective against Ebola virus have been mapped and in non-human primates CTL-mediated protection between viral strains (John Dye: specify) has been demonstrated using two filoviral proteins, nucleoprotein (NP) and glycoprotein (GP). These immunological results suggestmore » that the CTL avenue of immunity deserves consideration for a vaccine. The poorly-understood viral reservoirs means that it is difficult to predict what strains are likely to cause epidemics. Thus, there is a premium on developing a pan-filoviral vaccine. The genetic diversity of FILV is large, roughly the same scale as human immunodeficiency virus (HIV). This presents a serious challenge for the vaccine designer because a traditional vaccine aspiring to pan-filoviral coverage is likely to require the inclusion of many antigenic reagents. A recent method for optimizing cytotoxic T-cell lymphocyte epitope coverage with mosaic antigens was successful in improving potential CTL epitope coverage against HIV and may be useful in the context of very different viruses, such as the filoviruses discussed here. Mosaic proteins are recombinants composed of fragments of wild-type proteins joined at locations resulting in exclusively natural k-mers, 9 {le} k {le} 15, and having approximately the same length as the wild-type proteins. The use of mosaic antigens is motivated by three conjectures: (1) optimizing a mosaic protein to maximize coverage of k-mers found in a set of reference proteins will give better odds of including broadly-protective CTL epitopes in a vaccine than is possible with a wild-type protein, (2) reducing the number of low-prevalence k-mers minimizes the likelihood of undesirable immunodominance, and (3) excluding exogenous k-mers will result in mosaic proteins whose processing for presentation is close to what occurs with wild-type proteins. The first and second applications of the mosaic method were to HIV and Hepatitis C Virus (HCV). HIV is the virus with the largest number of known sequences, and consequently a plethora of information for the CTL vaccine designer to incorporate into their mosaics. Experience with HIV and HCV mosaics supports the validity of the three conjectures above. The available FILV sequences are probably closer to the minimum amount of information needed to make a meaningful mosaic vaccine candidate. There were 532 protein sequences in the National Institutes of Health GenPept database in November 2007 when our reference set was downloaded. These sequences come from both Ebola and Marburg viruses (EBOV and MARV), representing transcripts of all 7 genes. The coverage of viral diversity by the 7 genes is variable, with genes 1 (nucleoprotein, NP), 4 (glycoprotein, GP; soluble glycoprotein, sGP) and 7 (polymerase, L) giving the best coverage. Broadly-protective vaccine candidates for diverse viruses, such as HIV or Hepatitis C virus (HCV) have required pools of antigens. FILV is similar in this regard. While we have designed CTL mosaic proteins using all 7 types of filoviral proteins, only NP, GP and L proteins are reported here. If it were important to include other proteins in a mosaic CTL vaccine, additional sequences would be required to cover the space of known viral diversity.« less
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Savidor, Alon; Barzilay, Rotem; Elinger, Dalia; Yarden, Yosef; Lindzen, Moshit; Gabashvili, Alexandra; Adiv Tal, Ophir; Levin, Yishai
2017-06-01
Traditional "bottom-up" proteomic approaches use proteolytic digestion, LC-MS/MS, and database searching to elucidate peptide identities and their parent proteins. Protein sequences absent from the database cannot be identified, and even if present in the database, complete sequence coverage is rarely achieved even for the most abundant proteins in the sample. Thus, sequencing of unknown proteins such as antibodies or constituents of metaproteomes remains a challenging problem. To date, there is no available method for full-length protein sequencing, independent of a reference database, in high throughput. Here, we present Database-independent Protein Sequencing, a method for unambiguous, rapid, database-independent, full-length protein sequencing. The method is a novel combination of non-enzymatic, semi-random cleavage of the protein, LC-MS/MS analysis, peptide de novo sequencing, extraction of peptide tags, and their assembly into a consensus sequence using an algorithm named "Peptide Tag Assembler." As proof-of-concept, the method was applied to samples of three known proteins representing three size classes and to a previously un-sequenced, clinically relevant monoclonal antibody. Excluding leucine/isoleucine and glutamic acid/deamidated glutamine ambiguities, end-to-end full-length de novo sequencing was achieved with 99-100% accuracy for all benchmarking proteins and the antibody light chain. Accuracy of the sequenced antibody heavy chain, including the entire variable region, was also 100%, but there was a 23-residue gap in the constant region sequence. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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NASA Astrophysics Data System (ADS)
Basyuni, M.; Sulistiyono, N.; Wati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.
2018-03-01
Cloning of Kandelia obovata KcCAS gene (previously known as Kandelia candel) and Rhizophora stylosa RsCAS have already have been reported and encoded cycloartenol synthases. In this study, the predicted KcCAS and RsCAS protein were analyzed using online software of Phyre2 and Swiss-model. The protein modelling for KcCAS and RsCAS cycloartenol synthases was determined using Pyre2 had similar results with slightly different in sequence identity. By contrast, the Swiss-model for KcCAS slightly had higher sequence identity (47.31%) and Qmean (0.70) compared to RsCAS. No difference of ligands binding site which is considered as modulators for both cycloartenol synthases. The range of predicted protein derived from 91-757 amino acid residues with coverage sequence similarities 0.86, respectively from template model of lanosterol synthase from the human. Homology modelling revealed that 706 residues (93% of the amino acid sequence) had been modelled with 100.0% confidence by the single highest scoring template for both KcCAS and RsCAS using Phyre2. This coverage was more elevated than swiss-model predicted (86%). The present study suggested that both genes are responsible for the genesis of cycloartenol in these mangrove plants.
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Arnold, Roland; Goldenberg, Florian; Mewes, Hans-Werner; Rattei, Thomas
2014-01-01
The Similarity Matrix of Proteins (SIMAP, http://mips.gsf.de/simap/) database has been designed to massively accelerate computationally expensive protein sequence analysis tasks in bioinformatics. It provides pre-calculated sequence similarities interconnecting the entire known protein sequence universe, complemented by pre-calculated protein features and domains, similarity clusters and functional annotations. SIMAP covers all major public protein databases as well as many consistently re-annotated metagenomes from different repositories. As of September 2013, SIMAP contains >163 million proteins corresponding to ∼70 million non-redundant sequences. SIMAP uses the sensitive FASTA search heuristics, the Smith–Waterman alignment algorithm, the InterPro database of protein domain models and the BLAST2GO functional annotation algorithm. SIMAP assists biologists by facilitating the interactive exploration of the protein sequence universe. Web-Service and DAS interfaces allow connecting SIMAP with any other bioinformatic tool and resource. All-against-all protein sequence similarity matrices of project-specific protein collections are generated on request. Recent improvements allow SIMAP to cover the rapidly growing sequenced protein sequence universe. New Web-Service interfaces enhance the connectivity of SIMAP. Novel tools for interactive extraction of protein similarity networks have been added. Open access to SIMAP is provided through the web portal; the portal also contains instructions and links for software access and flat file downloads. PMID:24165881
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Wongkongkathep, Piriya; Li, Huilin; Zhang, Xing; Loo, Rachel R Ogorzalek; Julian, Ryan R; Loo, Joseph A
2015-11-15
The application of ion pre-activation with 266 nm ultraviolet (UV) laser irradiation combined with electron capture dissociation (ECD) is demonstrated to enhance top-down mass spectrometry sequence coverage of disulfide bond containing proteins. UV-based activation can homolytically cleave a disulfide bond to yield two separated thiol radicals. Activated ECD experiments of insulin and ribonuclease A containing three and four disulfide bonds, respectively, were performed. UV-activation in combination with ECD allowed the three disulfide bonds of insulin to be cleaved and the overall sequence coverage to be increased. For the larger sized ribonuclease A with four disulfide bonds, irradiation from an infrared laser (10.6 µm) to disrupt non-covalent interactions was combined with UV-activation to facilitate the cleavage of up to three disulfide bonds. Preferences for disulfide bond cleavage are dependent on protein structure and sequence. Disulfide bonds can reform if the generated radicals remain in close proximity. By varying the time delay between the UV-activation and the ECD events, it was determined that disulfide bonds reform within 10-100 msec after their UV-homolytic cleavage.
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Mowlaboccus, Shakeel; Perkins, Timothy T.; Smith, Helen; Sloots, Theo; Tozer, Sarah; Prempeh, Lydia-Jessica; Tay, Chin Yen; Peters, Fanny; Speers, David; Keil, Anthony D.; Kahler, Charlene M.
2016-01-01
Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). The BEXSERO® vaccine which is used to prevent serogroup B disease is composed of four sub-capsular protein antigens supplemented with an outer membrane vesicle. Since the sub-capsular protein antigens are variably expressed and antigenically variable amongst meningococcal isolates, vaccine coverage can be estimated by the meningococcal antigen typing system (MATS) which measures the propensity of the strain to be killed by vaccinated sera. Whole genome sequencing (WGS) which identifies the alleles of the antigens that may be recognised by the antibody response could represent, in future, an alternative estimate of coverage. In this study, WGS of 278 meningococcal isolates responsible for 62% of IMD in Western Australia from 2000–2014 were analysed for association of genetic lineage (sequence type [ST], clonal complex [cc]) with BEXSERO® antigen sequence type (BAST) and MATS to predict the annual vaccine coverage. A hyper-endemic period of IMD between 2000–05 was caused by cc41/44 with the major sequence type of ST-146 which was not predicted by MATS or BAST to be covered by the vaccine. An increase in serogroup diversity was observed between 2010–14 with the emergence of cc11 serogroup W in the adolescent population and cc23 serogroup Y in the elderly. BASTs were statistically associated with clonal complex although individual antigens underwent antigenic drift from the major type. BAST and MATS predicted an annual range of 44–91% vaccine coverage. Periods of low vaccine coverage in years post-2005 were not a result of the resurgence of cc41/44:ST-146 but were characterised by increased diversity of clonal complexes expressing BASTs which were not predicted by MATS to be covered by the vaccine. The driving force behind the diversity of the clonal complex and BAST during these periods of low vaccine coverage is unknown, but could be due to immune selection and inter-strain competition with carriage of non-disease causing meningococci. PMID:27355628
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Guan, Xiaoyan; Brownstein, Naomi C; Young, Nicolas L; Marshall, Alan G
2017-01-30
Bottom-up tandem mass spectrometry (MS/MS) is regularly used in proteomics to identify proteins from a sequence database. De novo sequencing is also available for sequencing peptides with relatively short sequence lengths. We recently showed that paired Lys-C and Lys-N proteases produce peptides of identical mass and similar retention time, but different tandem mass spectra. Such parallel experiments provide complementary information, and allow for up to 100% MS/MS sequence coverage. Here, we report digestion by paired Lys-C and Lys-N proteases of a seven-protein mixture: human hemoglobin alpha, bovine carbonic anhydrase 2, horse skeletal muscle myoglobin, hen egg white lysozyme, bovine pancreatic ribonuclease, bovine rhodanese, and bovine serum albumin, followed by reversed-phase nanoflow liquid chromatography, collision-induced dissociation, and 14.5 T Fourier transform ion cyclotron resonance mass spectrometry. Matched pairs of product peptide ions of equal precursor mass and similar retention times from each digestion are compared, leveraging single-residue transposed information with independent interferences to confidently identify fragment ion types, residues, and peptides. Selected pairs of product ion mass spectra for de novo sequenced protein segments from each member of the mixture are presented. Pairs of the transposed product ions as well as complementary information from the parallel experiments allow for both high MS/MS coverage for long peptide sequences and high confidence in the amino acid identification. Moreover, the parallel experiments in the de novo sequencing reduce false-positive matches of product ions from the single-residue transposed peptides from the same segment, and thereby further improve the confidence in protein identification. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
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The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads.
Wang, Zhiwen; Hobson, Neil; Galindo, Leonardo; Zhu, Shilin; Shi, Daihu; McDill, Joshua; Yang, Linfeng; Hawkins, Simon; Neutelings, Godfrey; Datla, Raju; Lambert, Georgina; Galbraith, David W; Grassa, Christopher J; Geraldes, Armando; Cronk, Quentin C; Cullis, Christopher; Dash, Prasanta K; Kumar, Polumetla A; Cloutier, Sylvie; Sharpe, Andrew G; Wong, Gane K-S; Wang, Jun; Deyholos, Michael K
2012-11-01
Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.
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Fujimori, Shigeo; Hirai, Naoya; Ohashi, Hiroyuki; Masuoka, Kazuyo; Nishikimi, Akihiko; Fukui, Yoshinori; Washio, Takanori; Oshikubo, Tomohiro; Yamashita, Tatsuhiro; Miyamoto-Sato, Etsuko
2012-01-01
Next-generation sequencing (NGS) has been applied to various kinds of omics studies, resulting in many biological and medical discoveries. However, high-throughput protein-protein interactome datasets derived from detection by sequencing are scarce, because protein-protein interaction analysis requires many cell manipulations to examine the interactions. The low reliability of the high-throughput data is also a problem. Here, we describe a cell-free display technology combined with NGS that can improve both the coverage and reliability of interactome datasets. The completely cell-free method gives a high-throughput and a large detection space, testing the interactions without using clones. The quantitative information provided by NGS reduces the number of false positives. The method is suitable for the in vitro detection of proteins that interact not only with the bait protein, but also with DNA, RNA and chemical compounds. Thus, it could become a universal approach for exploring the large space of protein sequences and interactome networks. PMID:23056904
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1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life
Mukherjee, Supratim; Seshadri, Rekha; Varghese, Neha J.; ...
2017-06-12
We present 1,003 reference genomes that were sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) initiative, selected to maximize sequence coverage of phylogenetic space. These genomes double the number of existing type strains and expand their overall phylogenetic diversity by 25%. Comparative analyses with previously available finished and draft genomes reveal a 10.5% increase in novel protein families as a function of phylogenetic diversity. The GEBA genomes recruit 25 million previously unassigned metagenomic proteins from 4,650 samples, improving their phylogenetic and functional interpretation. We identify numerous biosynthetic clusters and experimentally validate a divergent phenazine cluster withmore » potential new chemical structure and antimicrobial activity. This Resource is the largest single release of reference genomes to date. Bacterial and archaeal isolate sequence space is still far from saturated, and future endeavors in this direction will continue to be a valuable resource for scientific discovery.« less
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1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life
DOE Office of Scientific and Technical Information (OSTI.GOV)
Mukherjee, Supratim; Seshadri, Rekha; Varghese, Neha J.
We present 1,003 reference genomes that were sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA) initiative, selected to maximize sequence coverage of phylogenetic space. These genomes double the number of existing type strains and expand their overall phylogenetic diversity by 25%. Comparative analyses with previously available finished and draft genomes reveal a 10.5% increase in novel protein families as a function of phylogenetic diversity. The GEBA genomes recruit 25 million previously unassigned metagenomic proteins from 4,650 samples, improving their phylogenetic and functional interpretation. We identify numerous biosynthetic clusters and experimentally validate a divergent phenazine cluster withmore » potential new chemical structure and antimicrobial activity. This Resource is the largest single release of reference genomes to date. Bacterial and archaeal isolate sequence space is still far from saturated, and future endeavors in this direction will continue to be a valuable resource for scientific discovery.« less
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An estimated 5% of new protein structures solved today represent a new Pfam family
DOE Office of Scientific and Technical Information (OSTI.GOV)
Mistry, Jaina; Kloppmann, Edda; Rost, Burkhard
2013-11-01
This study uses the Pfam database to show that the sequence redundancy of protein structures deposited in the PDB is increasing. The possible reasons behind this trend are discussed. High-resolution structural knowledge is key to understanding how proteins function at the molecular level. The number of entries in the Protein Data Bank (PDB), the repository of all publicly available protein structures, continues to increase, with more than 8000 structures released in 2012 alone. The authors of this article have studied how structural coverage of the protein-sequence space has changed over time by monitoring the number of Pfam families that acquiredmore » their first representative structure each year from 1976 to 2012. Twenty years ago, for every 100 new PDB entries released, an estimated 20 Pfam families acquired their first structure. By 2012, this decreased to only about five families per 100 structures. The reasons behind the slower pace at which previously uncharacterized families are being structurally covered were investigated. It was found that although more than 50% of current Pfam families are still without a structural representative, this set is enriched in families that are small, functionally uncharacterized or rich in problem features such as intrinsically disordered and transmembrane regions. While these are important constraints, the reasons why it may not yet be time to give up the pursuit of a targeted but more comprehensive structural coverage of the protein-sequence space are discussed.« less
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Dessimoz, Christophe; Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro
2011-09-01
Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references.
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Zoller, Stefan; Manousaki, Tereza; Qiu, Huan; Meyer, Axel; Kuraku, Shigehiro
2011-01-01
Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references. PMID:21712341
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Caititu: a tool to graphically represent peptide sequence coverage and domain distribution.
Carvalho, Paulo C; Junqueira, Magno; Valente, Richard H; Domont, Gilberto B
2008-10-07
Here we present Caititu, an easy-to-use proteomics software to graphically represent peptide sequence coverage and domain distribution for different correlated samples (e.g. originated from 2D gel spots) relatively to the full-sequence of the known protein they are related to. Although Caititu has a broad applicability, we exemplify its usefulness in Toxinology using snake venom as a model. For example, proteolytic processing may lead to inactivation or loss of domains. Therefore, our proposed graphic representation for peptides identified by two dimensional electrophoresis followed by mass spectrometric identification of excised spots can aid in inferring what kind of processing happened to the toxins, if any. Caititu is freely available to download at: http://pcarvalho.com/things/caititu.
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Comprehensive Analysis of Protein Modifications by Top-down Mass Spectrometry
Zhang, Han; Ge, Ying
2012-01-01
Mass spectrometry (MS)-based proteomics is playing an increasingly important role in cardiovascular research. Proteomics includes not only identification and quantification of proteins, but also the characterization of protein modifications such as post-translational modifications and sequence variants. The conventional bottom-up approach, involving proteolytic digestion of proteins into small peptides prior to MS analysis, is routinely used for protein identification and quantification with high throughput and automation. Nevertheless, it has limitations in the analysis of protein modifications mainly due to the partial sequence coverage and loss of connections among modifications on disparate portions of a protein. An alternative approach, top-down MS, has emerged as a powerful tool for the analysis of protein modifications. The top-down approach analyzes whole proteins directly, providing a “bird’s eye” view of all existing modifications. Subsequently, each modified protein form can be isolated and fragmented in the mass spectrometer to locate the modification site. The incorporation of the non-ergodic dissociation methods such as electron capture dissociation (ECD) greatly enhances the top-down capabilities. ECD is especially useful for mapping labile post-translational modifications which are well-preserved during the ECD fragmentation process. Top-down MS with ECD has been successfully applied to cardiovascular research with the unique advantages in unraveling the molecular complexity, quantifying modified protein forms, complete mapping of modifications with full sequence coverage, discovering unexpected modifications, and identifying and quantifying positional isomers and determining the order of multiple modifications. Nevertheless, top-down MS still needs to overcome some technical challenges to realize its full potential. Herein, we reviewed the advantages and challenges of top-down methodology with a focus on its application in cardiovascular research. PMID:22187450
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Darville, Lancia N F; Merchant, Mark E; Maccha, Venkata; Siddavarapu, Vivekananda Reddy; Hasan, Azeem; Murray, Kermit K
2012-02-01
Mass spectrometry in conjunction with de novo sequencing was used to determine the amino acid sequence of a 35kDa lectin protein isolated from the serum of the American alligator that exhibits binding to mannose. The protein N-terminal sequence was determined using Edman degradation and enzymatic digestion with different proteases was used to generate peptide fragments for analysis by liquid chromatography tandem mass spectrometry (LC MS/MS). Separate analysis of the protein digests with multiple enzymes enhanced the protein sequence coverage. De novo sequencing was accomplished using MASCOT Distiller and PEAKS software and the sequences were searched against the NCBI database using MASCOT and BLAST to identify homologous peptides. MS analysis of the intact protein indicated that it is present primarily as monomer and dimer in vitro. The isolated 35kDa protein was ~98% sequenced and found to have 313 amino acids and nine cysteine residues and was identified as an alligator lectin. The alligator lectin sequence was aligned with other lectin sequences using DIALIGN and ClustalW software and was found to exhibit 58% and 59% similarity to both human and mouse intelectin-1. The alligator lectin exhibited strong binding affinities toward mannan and mannose as compared to other tested carbohydrates. Copyright © 2011 Elsevier Inc. All rights reserved.
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Reiz, Bela; Li, Liang
2010-09-01
Controlled hydrolysis of proteins to generate peptide ladders combined with mass spectrometric analysis of the resultant peptides can be used for protein sequencing. In this paper, two methods of improving the microwave-assisted protein hydrolysis process are described to enable rapid sequencing of proteins containing disulfide bonds and increase sequence coverage, respectively. It was demonstrated that proteins containing disulfide bonds could be sequenced by MS analysis by first performing hydrolysis for less than 2 min, followed by 1 h of reduction to release the peptides originally linked by disulfide bonds. It was shown that a strong base could be used as a catalyst for microwave-assisted protein hydrolysis, producing complementary sequence information to that generated by microwave-assisted acid hydrolysis. However, using either acid or base hydrolysis, amide bond breakages in small regions of the polypeptide chains of the model proteins (e.g., cytochrome c and lysozyme) were not detected. Dynamic light scattering measurement of the proteins solubilized in an acid or base indicated that protein-protein interaction or aggregation was not the cause of the failure to hydrolyze certain amide bonds. It was speculated that there were some unknown local structures that might play a role in preventing an acid or base from reacting with the peptide bonds therein. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.
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Virome Assembly and Annotation: A Surprise in the Namib Desert
Hesse, Uljana; van Heusden, Peter; Kirby, Bronwyn M.; Olonade, Israel; van Zyl, Leonardo J.; Trindade, Marla
2017-01-01
Sequencing, assembly, and annotation of environmental virome samples is challenging. Methodological biases and differences in species abundance result in fragmentary read coverage; sequence reconstruction is further complicated by the mosaic nature of viral genomes. In this paper, we focus on biocomputational aspects of virome analysis, emphasizing latent pitfalls in sequence annotation. Using simulated viromes that mimic environmental data challenges we assessed the performance of five assemblers (CLC-Workbench, IDBA-UD, SPAdes, RayMeta, ABySS). Individual analyses of relevant scaffold length fractions revealed shortcomings of some programs in reconstruction of viral genomes with excessive read coverage (IDBA-UD, RayMeta), and in accurate assembly of scaffolds ≥50 kb (SPAdes, RayMeta, ABySS). The CLC-Workbench assembler performed best in terms of genome recovery (including highly covered genomes) and correct reconstruction of large scaffolds; and was used to assemble a virome from a copper rich site in the Namib Desert. We found that scaffold network analysis and cluster-specific read reassembly improved reconstruction of sequences with excessive read coverage, and that strict data filtering for non-viral sequences prior to downstream analyses was essential. In this study we describe novel viral genomes identified in the Namib Desert copper site virome. Taxonomic affiliations of diverse proteins in the dataset and phylogenetic analyses of circovirus-like proteins indicated links to the marine habitat. Considering additional evidence from this dataset we hypothesize that viruses may have been carried from the Atlantic Ocean into the Namib Desert by fog and wind, highlighting the impact of the extended environment on an investigated niche in metagenome studies. PMID:28167933
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Phylogenomics from Whole Genome Sequences Using aTRAM.
Allen, Julie M; Boyd, Bret; Nguyen, Nam-Phuong; Vachaspati, Pranjal; Warnow, Tandy; Huang, Daisie I; Grady, Patrick G S; Bell, Kayce C; Cronk, Quentin C B; Mugisha, Lawrence; Pittendrigh, Barry R; Leonardi, M Soledad; Reed, David L; Johnson, Kevin P
2017-09-01
Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes ($<$1000 Mbp) it is feasible to sequence the entire genome at modest coverage ($10-30\\times$). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single-copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out-groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close- to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above $5-10\\times$, aTRAM was successful at assembling 80-90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.]. © The Author(s) 2017. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
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Graph-based optimization of epitope coverage for vaccine antigen design
Theiler, James Patrick; Korber, Bette Tina Marie
2017-01-29
Epigraph is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population. Potential epitopes are defined as short contiguous stretches of proteins, comparable in length to T-cell epitopes. This optimal coverage problem can be formulated in terms of a directed graph, with candidate antigens represented as paths that traverse this graph. Epigraph protein sequences can also be used as the basis for designing peptides for experimental evaluation of immune responses in natural infections to highly variable proteins. The epigraph tool suite also enables rapidmore » characterization of populations of diverse sequences from an immunological perspective. Fundamental distance measures are based on immunologically relevant shared potential epitope frequencies, rather than simple Hamming or phylogenetic distances. Here, we provide a mathematical description of the epigraph algorithm, include a comparison of different heuristics that can be used when graphs are not acyclic, and we describe an additional tool we have added to the web-based epigraph tool suite that provides frequency summaries of all distinct potential epitopes in a population. Lastly, we also show examples of the graphical output and summary tables that can be generated using the epigraph tool suite and explain their content and applications.« less
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Graph-based optimization of epitope coverage for vaccine antigen design
DOE Office of Scientific and Technical Information (OSTI.GOV)
Theiler, James Patrick; Korber, Bette Tina Marie
Epigraph is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population. Potential epitopes are defined as short contiguous stretches of proteins, comparable in length to T-cell epitopes. This optimal coverage problem can be formulated in terms of a directed graph, with candidate antigens represented as paths that traverse this graph. Epigraph protein sequences can also be used as the basis for designing peptides for experimental evaluation of immune responses in natural infections to highly variable proteins. The epigraph tool suite also enables rapidmore » characterization of populations of diverse sequences from an immunological perspective. Fundamental distance measures are based on immunologically relevant shared potential epitope frequencies, rather than simple Hamming or phylogenetic distances. Here, we provide a mathematical description of the epigraph algorithm, include a comparison of different heuristics that can be used when graphs are not acyclic, and we describe an additional tool we have added to the web-based epigraph tool suite that provides frequency summaries of all distinct potential epitopes in a population. Lastly, we also show examples of the graphical output and summary tables that can be generated using the epigraph tool suite and explain their content and applications.« less
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Brodie, Nicholas I; Huguet, Romain; Zhang, Terry; Viner, Rosa; Zabrouskov, Vlad; Pan, Jingxi; Petrotchenko, Evgeniy V; Borchers, Christoph H
2018-03-06
Top-down hydrogen-deuterium exchange (HDX) analysis using electron capture or transfer dissociation Fourier transform mass spectrometry (FTMS) is a powerful method for the analysis of secondary structure of proteins in solution. The resolution of the method is a function of the degree of fragmentation of backbone bonds in the proteins. While fragmentation is usually extensive near the N- and C-termini, electron capture (ECD) or electron transfer dissociation (ETD) fragmentation methods sometimes lack good coverage of certain regions of the protein, most often in the middle of the sequence. Ultraviolet photodissociation (UVPD) is a recently developed fast-fragmentation technique, which provides extensive backbone fragmentation that can be complementary in sequence coverage to the aforementioned electron-based fragmentation techniques. Here, we explore the application of electrospray ionization (ESI)-UVPD FTMS on an Orbitrap Fusion Lumos Tribrid mass spectrometer to top-down HDX analysis of proteins. We have incorporated UVPD-specific fragment-ion types and fragment-ion mixtures into our isotopic envelope fitting software (HDX Match) for the top-down HDX analysis. We have shown that UVPD data is complementary to ETD, thus improving the overall resolution when used as a combined approach.
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Yefremova, Yelena; Al-Majdoub, Mahmoud; Opuni, Kwabena F M; Koy, Cornelia; Cui, Weidong; Yan, Yuetian; Gross, Michael L; Glocker, Michael O
2015-03-01
Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.
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Dang, Xibei; Young, Nicolas L
2014-05-01
Ultraviolet photodissociation (UVPD) is a compelling fragmentation technique with great potential to enhance proteomics generally and top-down MS specifically. In this issue, Cannon et al. (Proteomics 2014, 14, XXXX-XXXX) use UVPD to perform top-down MS on several sequence variants of green fluorescent protein and compare the results to CID, higher energy collision induced dissociation, and electron transfer dissociation. As compared to the other techniques UVPD produces a wider variety of fragment ion types that are relatively evenly distributed across the protein sequences. Overall, their results demonstrate enhanced sequence coverage and higher confidence in sequence assignment via UVPD MS. Based on these and other recent results UVPD is certain to become an increasingly widespread and valuable tool for top-down proteomics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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The standard operating procedure of the DOE-JGI Metagenome Annotation Pipeline (MAP v.4)
Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos; ...
2016-02-24
The DOE-JGI Metagenome Annotation Pipeline (MAP v.4) performs structural and functional annotation for metagenomic sequences that are submitted to the Integrated Microbial Genomes with Microbiomes (IMG/M) system for comparative analysis. The pipeline runs on nucleotide sequences provide d via the IMG submission site. Users must first define their analysis projects in GOLD and then submit the associated sequence datasets consisting of scaffolds/contigs with optional coverage information and/or unassembled reads in fasta and fastq file formats. The MAP processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNAs, as well as CRISPR elements. Structural annotation ismore » followed by functional annotation including assignment of protein product names and connection to various protein family databases.« less
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The standard operating procedure of the DOE-JGI Metagenome Annotation Pipeline (MAP v.4)
DOE Office of Scientific and Technical Information (OSTI.GOV)
Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos
The DOE-JGI Metagenome Annotation Pipeline (MAP v.4) performs structural and functional annotation for metagenomic sequences that are submitted to the Integrated Microbial Genomes with Microbiomes (IMG/M) system for comparative analysis. The pipeline runs on nucleotide sequences provide d via the IMG submission site. Users must first define their analysis projects in GOLD and then submit the associated sequence datasets consisting of scaffolds/contigs with optional coverage information and/or unassembled reads in fasta and fastq file formats. The MAP processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNAs, as well as CRISPR elements. Structural annotation ismore » followed by functional annotation including assignment of protein product names and connection to various protein family databases.« less
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The Protein Information Resource: an integrated public resource of functional annotation of proteins
Wu, Cathy H.; Huang, Hongzhan; Arminski, Leslie; Castro-Alvear, Jorge; Chen, Yongxing; Hu, Zhang-Zhi; Ledley, Robert S.; Lewis, Kali C.; Mewes, Hans-Werner; Orcutt, Bruce C.; Suzek, Baris E.; Tsugita, Akira; Vinayaka, C. R.; Yeh, Lai-Su L.; Zhang, Jian; Barker, Winona C.
2002-01-01
The Protein Information Resource (PIR) serves as an integrated public resource of functional annotation of protein data to support genomic/proteomic research and scientific discovery. The PIR, in collaboration with the Munich Information Center for Protein Sequences (MIPS) and the Japan International Protein Information Database (JIPID), produces the PIR-International Protein Sequence Database (PSD), the major annotated protein sequence database in the public domain, containing about 250 000 proteins. To improve protein annotation and the coverage of experimentally validated data, a bibliography submission system is developed for scientists to submit, categorize and retrieve literature information. Comprehensive protein information is available from iProClass, which includes family classification at the superfamily, domain and motif levels, structural and functional features of proteins, as well as cross-references to over 40 biological databases. To provide timely and comprehensive protein data with source attribution, we have introduced a non-redundant reference protein database, PIR-NREF. The database consists of about 800 000 proteins collected from PIR-PSD, SWISS-PROT, TrEMBL, GenPept, RefSeq and PDB, with composite protein names and literature data. To promote database interoperability, we provide XML data distribution and open database schema, and adopt common ontologies. The PIR web site (http://pir.georgetown.edu/) features data mining and sequence analysis tools for information retrieval and functional identification of proteins based on both sequence and annotation information. The PIR databases and other files are also available by FTP (ftp://nbrfa.georgetown.edu/pir_databases). PMID:11752247
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Design and Initial Characterization of the SC-200 Proteomics Standard Mixture
Bauman, Andrew; Higdon, Roger; Rapson, Sean; Loiue, Brenton; Hogan, Jason; Stacy, Robin; Napuli, Alberto; Guo, Wenjin; van Voorhis, Wesley; Roach, Jared; Lu, Vincent; Landorf, Elizabeth; Stewart, Elizabeth; Kolker, Natali; Collart, Frank; Myler, Peter; van Belle, Gerald
2011-01-01
Abstract High-throughput (HTP) proteomics studies generate large amounts of data. Interpretation of these data requires effective approaches to distinguish noise from biological signal, particularly as instrument and computational capacity increase and studies become more complex. Resolving this issue requires validated and reproducible methods and models, which in turn requires complex experimental and computational standards. The absence of appropriate standards and data sets for validating experimental and computational workflows hinders the development of HTP proteomics methods. Most protein standards are simple mixtures of proteins or peptides, or undercharacterized reference standards in which the identity and concentration of the constituent proteins is unknown. The Seattle Children's 200 (SC-200) proposed proteomics standard mixture is the next step toward developing realistic, fully characterized HTP proteomics standards. The SC-200 exhibits a unique modular design to extend its functionality, and consists of 200 proteins of known identities and molar concentrations from 6 microbial genomes, distributed into 10 molar concentration tiers spanning a 1,000-fold range. We describe the SC-200's design, potential uses, and initial characterization. We identified 84% of SC-200 proteins with an LTQ-Orbitrap and 65% with an LTQ-Velos (false discovery rate = 1% for both). There were obvious trends in success rate, sequence coverage, and spectral counts with protein concentration; however, protein identification, sequence coverage, and spectral counts vary greatly within concentration levels. PMID:21250827
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Design and initial characterization of the SC-200 proteomics standard mixture.
Bauman, Andrew; Higdon, Roger; Rapson, Sean; Loiue, Brenton; Hogan, Jason; Stacy, Robin; Napuli, Alberto; Guo, Wenjin; van Voorhis, Wesley; Roach, Jared; Lu, Vincent; Landorf, Elizabeth; Stewart, Elizabeth; Kolker, Natali; Collart, Frank; Myler, Peter; van Belle, Gerald; Kolker, Eugene
2011-01-01
High-throughput (HTP) proteomics studies generate large amounts of data. Interpretation of these data requires effective approaches to distinguish noise from biological signal, particularly as instrument and computational capacity increase and studies become more complex. Resolving this issue requires validated and reproducible methods and models, which in turn requires complex experimental and computational standards. The absence of appropriate standards and data sets for validating experimental and computational workflows hinders the development of HTP proteomics methods. Most protein standards are simple mixtures of proteins or peptides, or undercharacterized reference standards in which the identity and concentration of the constituent proteins is unknown. The Seattle Children's 200 (SC-200) proposed proteomics standard mixture is the next step toward developing realistic, fully characterized HTP proteomics standards. The SC-200 exhibits a unique modular design to extend its functionality, and consists of 200 proteins of known identities and molar concentrations from 6 microbial genomes, distributed into 10 molar concentration tiers spanning a 1,000-fold range. We describe the SC-200's design, potential uses, and initial characterization. We identified 84% of SC-200 proteins with an LTQ-Orbitrap and 65% with an LTQ-Velos (false discovery rate = 1% for both). There were obvious trends in success rate, sequence coverage, and spectral counts with protein concentration; however, protein identification, sequence coverage, and spectral counts vary greatly within concentration levels.
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Application of Tandem Two-Dimensional Mass Spectrometry for Top-Down Deep Sequencing of Calmodulin
NASA Astrophysics Data System (ADS)
Floris, Federico; Chiron, Lionel; Lynch, Alice M.; Barrow, Mark P.; Delsuc, Marc-André; O'Connor, Peter B.
2018-06-01
Two-dimensional mass spectrometry (2DMS) involves simultaneous acquisition of the fragmentation patterns of all the analytes in a mixture by correlating their precursor and fragment ions by modulating precursor ions systematically through a fragmentation zone. Tandem two-dimensional mass spectrometry (MS/2DMS) unites the ultra-high accuracy of Fourier transform ion cyclotron resonance (FT-ICR) MS/MS and the simultaneous data-independent fragmentation of 2DMS to achieve extensive inter-residue fragmentation of entire proteins. 2DMS was recently developed for top-down proteomics (TDP), and applied to the analysis of calmodulin (CaM), reporting a cleavage coverage of about 23% using infrared multiphoton dissociation (IRMPD) as fragmentation technique. The goal of this work is to expand the utility of top-down protein analysis using MS/2DMS in order to extend the cleavage coverage in top-down proteomics further into the interior regions of the protein. In this case, using MS/2DMS, the cleavage coverage of CaM increased from 23% to 42%.
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DeHart, Caroline J.; Schweitzer, Mary H.; Thomas, Paul M.; Kelleher, Neil L.
2016-01-01
Proteomic studies of bone require specialized extraction protocols to demineralize and solubilize proteins from within the bone matrix. Although various protocols exist for bone protein recovery, little is known about how discrete steps in each protocol affect the subset of the bone proteome recovered by mass spectrometry (MS) analyses. Characterizing these different “extractomes” will provide critical data for development of novel and more efficient protein extraction methodologies for fossils. Here, we analyze 22 unique sub-extractions of chicken bone and directly compare individual extraction components for their total protein yield and diversity and coverage of bone proteins identified by MS. We extracted proteins using different combinations and ratios of demineralizing reagents, protein-solubilizing reagents, and post-extraction buffer removal methods, then evaluated tryptic digests from 20 µg aliquots of each fraction by tandem MS/MS on a 12T FT-ICR mass spectrometer. We compared total numbers of peptide spectral matches, peptides, and proteins identified from each fraction, the redundancy of protein identifications between discrete steps of extraction methods, and the sequence coverage obtained for select, abundant proteins. Although both alpha chains of collagen I (the most abundant protein in bone) were found in all fractions, other collagenous and non-collagenous proteins (e.g., apolipoprotein, osteonectin, hemoglobin) were differentially identified. We found that when a standardized amount of extracted proteins was analyzed, extraction steps that yielded the most protein (by weight) from bone were often not the ones that produced the greatest diversity of bone proteins, or the highest degree of protein coverage. Generally, the highest degrees of diversity and coverage were obtained from demineralization fractions, and the proteins found in the subsequent solubilization fractions were highly redundant with those in the previous fraction. Based on these data, we identify future directions and parameters to consider (e.g., proteins targeted, amount of sample required) when applying discrete parts of these protocols to fossils. PMID:27812413
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Identification of proteomic signatures associated with lung cancer and COPD.
Pastor, M D; Nogal, A; Molina-Pinelo, S; Meléndez, R; Salinas, A; González De la Peña, M; Martín-Juan, J; Corral, J; García-Carbonero, R; Carnero, A; Paz-Ares, L
2013-08-26
Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC. The aim of this study was to identify distinct proteomic profiles able to discriminate these two pathological entities. Protein content was assessed in the bronchoalveolar lavage (BAL) of 60 patients classified in four groups: COPD, COPD and LC, LC without COPD, and control with neither COPD nor LC. Proteins were separated into spots by bidimensional polyacrylamide gel electrophoresis (2D-PAGE) and examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF). A total of 40 proteins were differentially expressed in the LC and/or COPD groups as compared with the control group. Distinct protein profiles were identified and validated for each pathological entity (LC and COPD). The main networks involved were related to inflammatory signalling, free radical scavenging and oxidative stress response, and glycolysis and gluconeogenesis pathways. The most relevant signalling link between LC and COPD was through the NF-κB pathway. In conclusion, the protein profiles identified contribute to elucidate the underlying pathogenic pathways of both diseases, and provide new tools of potential use as biomarkers for the early diagnosis of LC. Sequence coverage. The protein sequence coverage (95%) was estimated for specific proteins by the percentage of matching amino acids from the identified peptides having confidence greater than or equal to 95% divided by the total number of amino acids in the sequence. Ingenuity Pathways Analysis. Mapping of our proteins onto biological pathways and disease networks demonstrated that 22 proteins were linked to inflammatory signalling (p-value: 1.35 10(-08)-1.42 10(-02)), 15 proteins were associated with free radical scavenging and oxidative stress response (p-value: 4.93 10(-11)-1.27 10(-02)), and 9 proteins were related with glycolysis and gluconeogenesis pathways (p-value: 7.39 10(-09)-1.58 10(-02)). Copyright © 2013 Elsevier B.V. All rights reserved.
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Detection of the HA-33 protein in botulinum neurotoxin type G complex by mass spectrometry.
Kalb, Suzanne R; Baudys, Jakub; Barr, John R
2015-10-23
The disease botulism is caused by intoxication with botulinum neurotoxins (BoNTs), extremely toxic proteins which cause paralysis. This neurotoxin is produced by some members of the Clostridium botulinum and closely related species, and is produced as a protein complex consisting of the neurotoxin and neurotoxin-associated proteins (NAPs). There are seven known serotypes of BoNT, A-G, and the composition of the NAPs can differ between these serotypes. It was previously published that the BoNT/G complex consisted of BoNT/G, nontoxic-nonhemagglutinin (NTNH), Hemagglutinin 70 (HA-70), and HA-17, but that HA-33, a component of the protein complex of other serotypes of BoNT, was not found. Components of the BoNT/G complex were first separated by SDS-PAGE, and bands corresponding to components of the complex were digested and analyzed by LC-MS/MS. Gel bands were identified with sequence coverages of 91% for BoNT/G, 91% for NTNH, 89% for HA-70, and 88% for HA-17. Notably, one gel band was also clearly identified as HA-33 with 93% sequence coverage. The BoNT/G complex consists of BoNT/G, NTNH, HA-70, HA-17, and HA-33. These proteins form the progenitor form of BoNT/G, similar to all other HA positive progenitor toxin complexes.
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Where Have All the Interactions Gone? Estimating the Coverage of Two-Hybrid Protein Interaction Maps
Huang, Hailiang; Jedynak, Bruno M; Bader, Joel S
2007-01-01
Yeast two-hybrid screens are an important method for mapping pairwise physical interactions between proteins. The fraction of interactions detected in independent screens can be very small, and an outstanding challenge is to determine the reason for the low overlap. Low overlap can arise from either a high false-discovery rate (interaction sets have low overlap because each set is contaminated by a large number of stochastic false-positive interactions) or a high false-negative rate (interaction sets have low overlap because each misses many true interactions). We extend capture–recapture theory to provide the first unified model for false-positive and false-negative rates for two-hybrid screens. Analysis of yeast, worm, and fly data indicates that 25% to 45% of the reported interactions are likely false positives. Membrane proteins have higher false-discovery rates on average, and signal transduction proteins have lower rates. The overall false-negative rate ranges from 75% for worm to 90% for fly, which arises from a roughly 50% false-negative rate due to statistical undersampling and a 55% to 85% false-negative rate due to proteins that appear to be systematically lost from the assays. Finally, statistical model selection conclusively rejects the Erdös-Rényi network model in favor of the power law model for yeast and the truncated power law for worm and fly degree distributions. Much as genome sequencing coverage estimates were essential for planning the human genome sequencing project, the coverage estimates developed here will be valuable for guiding future proteomic screens. All software and datasets are available in Datasets S1 and S2, Figures S1–S5, and Tables S1−S6, and are also available from our Web site, http://www.baderzone.org. PMID:18039026
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Zhao, Panpan; Zhong, Jiayong; Liu, Wanting; Zhao, Jing; Zhang, Gong
2017-12-01
Multiple search engines based on various models have been developed to search MS/MS spectra against a reference database, providing different results for the same data set. How to integrate these results efficiently with minimal compromise on false discoveries is an open question due to the lack of an independent, reliable, and highly sensitive standard. We took the advantage of the translating mRNA sequencing (RNC-seq) result as a standard to evaluate the integration strategies of the protein identifications from various search engines. We used seven mainstream search engines (Andromeda, Mascot, OMSSA, X!Tandem, pFind, InsPecT, and ProVerB) to search the same label-free MS data sets of human cell lines Hep3B, MHCCLM3, and MHCC97H from the Chinese C-HPP Consortium for Chromosomes 1, 8, and 20. As expected, the union of seven engines resulted in a boosted false identification, whereas the intersection of seven engines remarkably decreased the identification power. We found that identifications of at least two out of seven engines resulted in maximizing the protein identification power while minimizing the ratio of suspicious/translation-supported identifications (STR), as monitored by our STR index, based on RNC-Seq. Furthermore, this strategy also significantly improves the peptides coverage of the protein amino acid sequence. In summary, we demonstrated a simple strategy to significantly improve the performance for shotgun mass spectrometry by protein-level integrating multiple search engines, maximizing the utilization of the current MS spectra without additional experimental work.
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Benson, Dennis A.; Karsch-Mizrachi, Ilene; Lipman, David J.; Ostell, James; Wheeler, David L.
2007-01-01
GenBank (R) is a comprehensive database that contains publicly available nucleotide sequences for more than 240 000 named organisms, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the web-based BankIt or standalone Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the EMBL Data Library in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through NCBI's retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI Homepage (). PMID:17202161
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The Histone Database: an integrated resource for histones and histone fold-containing proteins
Mariño-Ramírez, Leonardo; Levine, Kevin M.; Morales, Mario; Zhang, Suiyuan; Moreland, R. Travis; Baxevanis, Andreas D.; Landsman, David
2011-01-01
Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins. Database URL: The Histone Sequence Database is freely available and can be accessed at http://research.nhgri.nih.gov/histones/. PMID:22025671
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Yuan, Huiming; Zhou, Yuan; Zhang, Lihua; Liang, Zhen; Zhang, Yukui
2009-10-30
An integrated platform with the combination of proteins and peptides separation was established via the unit of on-line proteins digestion, by which proteins were in sequence separated by column switch recycling size exclusion chromatography (csrSEC), on-line digested by an immobilized trypsin microreactor, trapped and desalted by two parallel C8 precolumns, separated by microRPLC with the linear gradient of organic modifier concentration, and identified by ESI-MS/MS. A 6-protein mixture, with Mr ranging from 10 kDa to 80 kDa, was used to evaluate the performance of the integrated platform, and all proteins were identified with sequence coverage over 5.67%. Our experimental results demonstrate that such an integrated platform is of advantages such as good time compatibility, high peak capacity, and facile automation, which might be a promising approach for proteome study.
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Li, Ying Hong; Xu, Jing Yu; Tao, Lin; Li, Xiao Feng; Li, Shuang; Zeng, Xian; Chen, Shang Ying; Zhang, Peng; Qin, Chu; Zhang, Cheng; Chen, Zhe; Zhu, Feng; Chen, Yu Zong
2016-01-01
Knowledge of protein function is important for biological, medical and therapeutic studies, but many proteins are still unknown in function. There is a need for more improved functional prediction methods. Our SVM-Prot web-server employed a machine learning method for predicting protein functional families from protein sequences irrespective of similarity, which complemented those similarity-based and other methods in predicting diverse classes of proteins including the distantly-related proteins and homologous proteins of different functions. Since its publication in 2003, we made major improvements to SVM-Prot with (1) expanded coverage from 54 to 192 functional families, (2) more diverse protein descriptors protein representation, (3) improved predictive performances due to the use of more enriched training datasets and more variety of protein descriptors, (4) newly integrated BLAST analysis option for assessing proteins in the SVM-Prot predicted functional families that were similar in sequence to a query protein, and (5) newly added batch submission option for supporting the classification of multiple proteins. Moreover, 2 more machine learning approaches, K nearest neighbor and probabilistic neural networks, were added for facilitating collective assessment of protein functions by multiple methods. SVM-Prot can be accessed at http://bidd2.nus.edu.sg/cgi-bin/svmprot/svmprot.cgi.
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Shirts, Brian H; Salipante, Stephen J; Casadei, Silvia; Ryan, Shawnia; Martin, Judith; Jacobson, Angela; Vlaskin, Tatyana; Koehler, Karen; Livingston, Robert J; King, Mary-Claire; Walsh, Tom; Pritchard, Colin C
2014-10-01
Single-exon inversions have rarely been described in clinical syndromes and are challenging to detect using Sanger sequencing. We report the case of a 40-year-old woman with adenomatous colon polyps too numerous to count and who had a complex inversion spanning the entire exon 10 in APC (the gene encoding for adenomatous polyposis coli), causing exon skipping and resulting in a frameshift and premature protein truncation. In this study, we employed complete APC gene sequencing using high-coverage next-generation sequencing by ColoSeq, analysis with BreakDancer and SLOPE software, and confirmatory transcript analysis. ColoSeq identified a complex small genomic rearrangement consisting of an inversion that results in translational skipping of exon 10 in the APC gene. This mutation would not have been detected by traditional sequencing or gene-dosage methods. We report a case of adenomatous polyposis resulting from a complex single-exon inversion. Our report highlights the benefits of large-scale sequencing methods that capture intronic sequences with high enough depth of coverage-as well as the use of informatics tools-to enable detection of small pathogenic structural rearrangements.
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Complete genome sequence of Menghai rhabdovirus, a novel mosquito-borne rhabdovirus from China.
Sun, Qiang; Zhao, Qiumin; An, Xiaoping; Guo, Xiaofang; Zuo, Shuqing; Zhang, Xianglilan; Pei, Guangqian; Liu, Wenli; Cheng, Shi; Wang, Yunfei; Shu, Peng; Mi, Zhiqiang; Huang, Yong; Zhang, Zhiyi; Tong, Yigang; Zhou, Hongning; Zhang, Jiusong
2017-04-01
Menghai rhabdovirus (MRV) was isolated from Aedes albopictus in Menghai county of Yunnan Province, China, in August 2010. Whole-genome sequencing of MRV was performed using an Ion PGM™ Sequencer. We found that MRV is a single-stranded, negative-sense RNA virus. The complete genome of MRV has 10,744 nt, with short inverted repeat termini, encoding five typical rhabdovirus proteins (N, P, M, G, and L) and an additional small hypothetical protein. Nucleotide BLAST analysis using the BLASTn method showed that the genome sequence most similar to that of MRV is that of Arboretum virus (NC_025393.1), with a Max score of 322, query coverage of 14%, and 66% identity. Genomic and phylogenetic analyses both demonstrated that MRV should be considered a member of a novel species of the family Rhabdoviridae.
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Mosaic protein and nucleic acid vaccines against hepatitis C virus
Yusim, Karina; Korber, Bette T. M.; Kuiken, Carla L.; Fischer, William M.
2013-06-11
The invention relates to immunogenic compositions useful as HCV vaccines. Provided are HCV mosaic polypeptide and nucleic acid compositions which provide higher levels of T-cell epitope coverage while minimizing the occurrence of unnatural and rare epitopes compared to natural HCV polypeptides and consensus HCV sequences.
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Covering complete proteomes with X-ray structures: A current snapshot
Mizianty, Marcin J.; Fan, Xiao; Yan, Jing; ...
2014-10-23
Structural genomics programs have developed and applied structure-determination pipelines to a wide range of protein targets, facilitating the visualization of macromolecular interactions and the understanding of their molecular and biochemical functions. The fundamental question of whether three-dimensional structures of all proteins and all functional annotations can be determined using X-ray crystallography is investigated. A first-of-its-kind large-scale analysis of crystallization propensity for all proteins encoded in 1953 fully sequenced genomes was performed. It is shown that current X-ray crystallographic knowhow combined with homology modeling can provide structures for 25% of modeling families (protein clusters for which structural models can be obtainedmore » through homology modeling), with at least one structural model produced for each Gene Ontology functional annotation. The coverage varies between superkingdoms, with 19% for eukaryotes, 35% for bacteria and 49% for archaea, and with those of viruses following the coverage values of their hosts. It is shown that the crystallization propensities of proteomes from the taxonomic superkingdoms are distinct. The use of knowledge-based target selection is shown to substantially increase the ability to produce X-ray structures. It is demonstrated that the human proteome has one of the highest attainable coverage values among eukaryotes, and GPCR membrane proteins suitable for X-ray structure determination were determined.« less
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Stable isotope, site-specific mass tagging for protein identification
Chen, Xian
2006-10-24
Proteolytic peptide mass mapping as measured by mass spectrometry provides an important method for the identification of proteins, which are usually identified by matching the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. The present invention describes a method for increasing the specificity, accuracy and efficiency of the assignments of particular proteolytic peptides and consequent protein identification, by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence without the need for ultrahigh instrumental accuracy. Selected amino acid(s) are labeled with .sup.13C/.sup.15N/.sup.2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tag(s) can then be readily distinguished from other peptides in mass spectra. The present method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency and accuracy for protein identifications.
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Du, Ruofei; Mercante, Donald; Fang, Zhide
2013-01-01
In functional metagenomics, BLAST homology search is a common method to classify metagenomic reads into protein/domain sequence families such as Clusters of Orthologous Groups of proteins (COGs) in order to quantify the abundance of each COG in the community. The resulting functional profile of the community is then used in downstream analysis to correlate the change in abundance to environmental perturbation, clinical variation, and so on. However, the short read length coupled with next-generation sequencing technologies poses a barrier in this approach, essentially because similarity significance cannot be discerned by searching with short reads. Consequently, artificial functional families are produced, in which those with a large number of reads assigned decreases the accuracy of functional profile dramatically. There is no method available to address this problem. We intended to fill this gap in this paper. We revealed that BLAST similarity scores of homologues for short reads from COG protein members coding sequences are distributed differently from the scores of those derived elsewhere. We showed that, by choosing an appropriate score cut-off, we are able to filter out most artificial families and simultaneously to preserve sufficient information in order to build the functional profile. We also showed that, by incorporated application of BLAST and RPS-BLAST, some artificial families with large read counts can be further identified after the score cutoff filtration. Evaluated on three experimental metagenomic datasets with different coverages, we found that the proposed method is robust against read coverage and consistently outperforms the other E-value cutoff methods currently used in literatures. PMID:23516532
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Whole Wiskott‑Aldrich syndrome protein gene deletion identified by high throughput sequencing.
He, Xiangling; Zou, Runying; Zhang, Bing; You, Yalan; Yang, Yang; Tian, Xin
2017-11-01
Wiskott‑Aldrich syndrome (WAS) is a rare X‑linked recessive immunodeficiency disorder, characterized by thrombocytopenia, small platelets, eczema and recurrent infections associated with increased risk of autoimmunity and malignancy disorders. Mutations in the WAS protein (WASP) gene are responsible for WAS. To date, WASP mutations, including missense/nonsense, splicing, small deletions, small insertions, gross deletions, and gross insertions have been identified in patients with WAS. In addition, WASP‑interacting proteins are suspected in patients with clinical features of WAS, in whom the WASP gene sequence and mRNA levels are normal. The present study aimed to investigate the application of next generation sequencing in definitive diagnosis and clinical therapy for WAS. A 5 month‑old child with WAS who displayed symptoms of thrombocytopenia was examined. Whole exome sequence analysis of genomic DNA showed that the coverage and depth of WASP were extremely low. Quantitative polymerase chain reaction indicated total WASP gene deletion in the proband. In conclusion, high throughput sequencing is useful for the verification of WAS on the genetic profile, and has implications for family planning guidance and establishment of clinical programs.
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Bang, Kyeongrin; Hwang, Sejung; Lee, Jiae; Cho, Saeyoull
2015-01-01
To identify immune-related genes in the larvae of white-spotted flower chafers, next-generation sequencing was conducted with an Illumina HiSeq2000, resulting in 100 million cDNA reads with sequence information from over 10 billion base pairs (bp) and >50× transcriptome coverage. A subset of 77,336 contigs was created, and ∼35,532 sequences matched entries against the NCBI nonredundant database (cutoff, e < 10(-5)). Statistical analysis was performed on the 35,532 contigs. For profiling of the immune response, samples were analyzed by aligning 42 base sequence tags to the de novo reference assembly, comparing levels in immunized larvae to control levels of expression. Of the differentially expressed genes, 3,440 transcripts were upregulated and 3,590 transcripts were downregulated. Many of these genes were confirmed as immune-related genes such as pattern recognition proteins, immune-related signal transduction proteins, antimicrobial peptides, and cellular response proteins, by comparison to published data. © The Author 2015. Published by Oxford University Press on behalf of the Entomological Society of America.
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Benson, Dennis A; Karsch-Mizrachi, Ilene; Lipman, David J; Ostell, James; Wheeler, David L
2008-01-01
GenBank (R) is a comprehensive database that contains publicly available nucleotide sequences for more than 260 000 named organisms, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the web-based BankIt or standalone Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the European Molecular Biology Laboratory Nucleotide Sequence Database in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through NCBI's retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI Homepage: www.ncbi.nlm.nih.gov.
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Benson, Dennis A.; Karsch-Mizrachi, Ilene; Lipman, David J.; Ostell, James; Wheeler, David L.
2008-01-01
GenBank (R) is a comprehensive database that contains publicly available nucleotide sequences for more than 260 000 named organisms, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the web-based BankIt or standalone Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the European Molecular Biology Laboratory Nucleotide Sequence Database in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through NCBI's retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI Homepage: www.ncbi.nlm.nih.gov PMID:18073190
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Gan, Han Ming; Tan, Mun Hua; Lee, Yin Peng; Austin, Christopher M
2016-05-01
The mitogenome of the Australian freshwater blackfish, Gadopsis marmoratus was recovered coverage by genome skimming using the MiSeq sequencer (GenBank Accession Number: NC_024436). The blackfish mitogenome has 16,407 base pairs made up of 13 protein-coding genes, 2 ribosomal subunit genes, 22 transfer RNAs, and a 819 bp non-coding AT-rich region. This is the 5th mitogenome sequence to be reported for the family Percichthyidae.
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Structural analysis of a set of proteins resulting from a bacterial genomics project.
Badger, J; Sauder, J M; Adams, J M; Antonysamy, S; Bain, K; Bergseid, M G; Buchanan, S G; Buchanan, M D; Batiyenko, Y; Christopher, J A; Emtage, S; Eroshkina, A; Feil, I; Furlong, E B; Gajiwala, K S; Gao, X; He, D; Hendle, J; Huber, A; Hoda, K; Kearins, P; Kissinger, C; Laubert, B; Lewis, H A; Lin, J; Loomis, K; Lorimer, D; Louie, G; Maletic, M; Marsh, C D; Miller, I; Molinari, J; Muller-Dieckmann, H J; Newman, J M; Noland, B W; Pagarigan, B; Park, F; Peat, T S; Post, K W; Radojicic, S; Ramos, A; Romero, R; Rutter, M E; Sanderson, W E; Schwinn, K D; Tresser, J; Winhoven, J; Wright, T A; Wu, L; Xu, J; Harris, T J R
2005-09-01
The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB. Copyright 2005 Wiley-Liss, Inc.
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Neumann, Sindy; Hartmann, Holger; Martin-Galiano, Antonio J; Fuchs, Angelika; Frishman, Dmitrij
2012-03-01
Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ∼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/. Copyright © 2011 Wiley Periodicals, Inc.
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Modularity of Protein Folds as a Tool for Template-Free Modeling of Structures.
Vallat, Brinda; Madrid-Aliste, Carlos; Fiser, Andras
2015-08-01
Predicting the three-dimensional structure of proteins from their amino acid sequences remains a challenging problem in molecular biology. While the current structural coverage of proteins is almost exclusively provided by template-based techniques, the modeling of the rest of the protein sequences increasingly require template-free methods. However, template-free modeling methods are much less reliable and are usually applicable for smaller proteins, leaving much space for improvement. We present here a novel computational method that uses a library of supersecondary structure fragments, known as Smotifs, to model protein structures. The library of Smotifs has saturated over time, providing a theoretical foundation for efficient modeling. The method relies on weak sequence signals from remotely related protein structures to create a library of Smotif fragments specific to the target protein sequence. This Smotif library is exploited in a fragment assembly protocol to sample decoys, which are assessed by a composite scoring function. Since the Smotif fragments are larger in size compared to the ones used in other fragment-based methods, the proposed modeling algorithm, SmotifTF, can employ an exhaustive sampling during decoy assembly. SmotifTF successfully predicts the overall fold of the target proteins in about 50% of the test cases and performs competitively when compared to other state of the art prediction methods, especially when sequence signal to remote homologs is diminishing. Smotif-based modeling is complementary to current prediction methods and provides a promising direction in addressing the structure prediction problem, especially when targeting larger proteins for modeling.
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Cuff, Alison L.; Sillitoe, Ian; Lewis, Tony; Clegg, Andrew B.; Rentzsch, Robert; Furnham, Nicholas; Pellegrini-Calace, Marialuisa; Jones, David; Thornton, Janet; Orengo, Christine A.
2011-01-01
CATH version 3.3 (class, architecture, topology, homology) contains 128 688 domains, 2386 homologous superfamilies and 1233 fold groups, and reflects a major focus on classifying structural genomics (SG) structures and transmembrane proteins, both of which are likely to add structural novelty to the database and therefore increase the coverage of protein fold space within CATH. For CATH version 3.4 we have significantly improved the presentation of sequence information and associated functional information for CATH superfamilies. The CATH superfamily pages now reflect both the functional and structural diversity within the superfamily and include structural alignments of close and distant relatives within the superfamily, annotated with functional information and details of conserved residues. A significantly more efficient search function for CATH has been established by implementing the search server Solr (http://lucene.apache.org/solr/). The CATH v3.4 webpages have been built using the Catalyst web framework. PMID:21097779
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Ciric, Milica; Moon, Christina D; Leahy, Sinead C; Creevey, Christopher J; Altermann, Eric; Attwood, Graeme T; Rakonjac, Jasna; Gagic, Dragana
2014-05-12
In silico, secretome proteins can be predicted from completely sequenced genomes using various available algorithms that identify membrane-targeting sequences. For metasecretome (collection of surface, secreted and transmembrane proteins from environmental microbial communities) this approach is impractical, considering that the metasecretome open reading frames (ORFs) comprise only 10% to 30% of total metagenome, and are poorly represented in the dataset due to overall low coverage of metagenomic gene pool, even in large-scale projects. By combining secretome-selective phage display and next-generation sequencing, we focused the sequence analysis of complex rumen microbial community on the metasecretome component of the metagenome. This approach achieved high enrichment (29 fold) of secreted fibrolytic enzymes from the plant-adherent microbial community of the bovine rumen. In particular, we identified hundreds of heretofore rare modules belonging to cellulosomes, cell-surface complexes specialised for recognition and degradation of the plant fibre. As a method, metasecretome phage display combined with next-generation sequencing has a power to sample the diversity of low-abundance surface and secreted proteins that would otherwise require exceptionally large metagenomic sequencing projects. As a resource, metasecretome display library backed by the dataset obtained by next-generation sequencing is ready for i) affinity selection by standard phage display methodology and ii) easy purification of displayed proteins as part of the virion for individual functional analysis.
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Gunawardena, Harsha P; O'Brien, Jonathon; Wrobel, John A; Xie, Ling; Davies, Sherri R; Li, Shunqiang; Ellis, Matthew J; Qaqish, Bahjat F; Chen, Xian
2016-02-01
Single quantitative platforms such as label-based or label-free quantitation (LFQ) present compromises in accuracy, precision, protein sequence coverage, and speed of quantifiable proteomic measurements. To maximize the quantitative precision and the number of quantifiable proteins or the quantifiable coverage of tissue proteomes, we have developed a unified approach, termed QuantFusion, that combines the quantitative ratios of all peptides measured by both LFQ and label-based methodologies. Here, we demonstrate the use of QuantFusion in determining the proteins differentially expressed in a pair of patient-derived tumor xenografts (PDXs) representing two major breast cancer (BC) subtypes, basal and luminal. Label-based in-spectra quantitative peptides derived from amino acid-coded tagging (AACT, also known as SILAC) of a non-malignant mammary cell line were uniformly added to each xenograft with a constant predefined ratio, from which Ratio-of-Ratio estimates were obtained for the label-free peptides paired with AACT peptides in each PDX tumor. A mixed model statistical analysis was used to determine global differential protein expression by combining complementary quantifiable peptide ratios measured by LFQ and Ratio-of-Ratios, respectively. With minimum number of replicates required for obtaining the statistically significant ratios, QuantFusion uses the distinct mechanisms to "rescue" the missing data inherent to both LFQ and label-based quantitation. Combined quantifiable peptide data from both quantitative schemes increased the overall number of peptide level measurements and protein level estimates. In our analysis of the PDX tumor proteomes, QuantFusion increased the number of distinct peptide ratios by 65%, representing differentially expressed proteins between the BC subtypes. This quantifiable coverage improvement, in turn, not only increased the number of measurable protein fold-changes by 8% but also increased the average precision of quantitative estimates by 181% so that some BC subtypically expressed proteins were rescued by QuantFusion. Thus, incorporating data from multiple quantitative approaches while accounting for measurement variability at both the peptide and global protein levels make QuantFusion unique for obtaining increased coverage and quantitative precision for tissue proteomes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Improved Analysis of Nanopore Sequence Data and Scanning Nanopore Techniques
NASA Astrophysics Data System (ADS)
Szalay, Tamas
The field of nanopore research has been driven by the need to inexpensively and rapidly sequence DNA. In order to help realize this goal, this thesis describes the PoreSeq algorithm that identifies and corrects errors in real-world nanopore sequencing data and improves the accuracy of de novo genome assembly with increasing coverage depth. The approach relies on modeling the possible sources of uncertainty that occur as DNA advances through the nanopore and then using this model to find the sequence that best explains multiple reads of the same region of DNA. PoreSeq increases nanopore sequencing read accuracy of M13 bacteriophage DNA from 85% to 99% at 100X coverage. We also use the algorithm to assemble E. coli with 30X coverage and the lambda genome at a range of coverages from 3X to 50X. Additionally, we classify sequence variants at an order of magnitude lower coverage than is possible with existing methods. This thesis also reports preliminary progress towards controlling the motion of DNA using two nanopores instead of one. The speed at which the DNA travels through the nanopore needs to be carefully controlled to facilitate the detection of individual bases. A second nanopore in close proximity to the first could be used to slow or stop the motion of the DNA in order to enable a more accurate readout. The fabrication process for a new pyramidal nanopore geometry was developed in order to facilitate the positioning of the nanopores. This thesis demonstrates that two of them can be placed close enough to interact with a single molecule of DNA, which is a prerequisite for being able to use the driving force of the pores to exert fine control over the motion of the DNA. Another strategy for reading the DNA is to trap it completely with one pore and to move the second nanopore instead. To that end, this thesis also shows that a single strand of immobilized DNA can be captured in a scanning nanopore and examined for a full hour, with data from many scans at many different voltages obtained in order to detect a bound protein placed partway along the molecule.
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Li, Chang-Lin; Li, Kai-Cheng; Wu, Dan; Chen, Yan; Luo, Hao; Zhao, Jing-Rong; Wang, Sa-Shuang; Sun, Ming-Ming; Lu, Ying-Jin; Zhong, Yan-Qing; Hu, Xu-Ye; Hou, Rui; Zhou, Bei-Bei; Bao, Lan; Xiao, Hua-Sheng; Zhang, Xu
2016-01-01
Sensory neurons are distinguished by distinct signaling networks and receptive characteristics. Thus, sensory neuron types can be defined by linking transcriptome-based neuron typing with the sensory phenotypes. Here we classify somatosensory neurons of the mouse dorsal root ganglion (DRG) by high-coverage single-cell RNA-sequencing (10 950 ± 1 218 genes per neuron) and neuron size-based hierarchical clustering. Moreover, single DRG neurons responding to cutaneous stimuli are recorded using an in vivo whole-cell patch clamp technique and classified by neuron-type genetic markers. Small diameter DRG neurons are classified into one type of low-threshold mechanoreceptor and five types of mechanoheat nociceptors (MHNs). Each of the MHN types is further categorized into two subtypes. Large DRG neurons are categorized into four types, including neurexophilin 1-expressing MHNs and mechanical nociceptors (MNs) expressing BAI1-associated protein 2-like 1 (Baiap2l1). Mechanoreceptors expressing trafficking protein particle complex 3-like and Baiap2l1-marked MNs are subdivided into two subtypes each. These results provide a new system for cataloging somatosensory neurons and their transcriptome databases. PMID:26691752
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Sequence Analysis of the Genome of Carnation (Dianthus caryophyllus L.)
Yagi, Masafumi; Kosugi, Shunichi; Hirakawa, Hideki; Ohmiya, Akemi; Tanase, Koji; Harada, Taro; Kishimoto, Kyutaro; Nakayama, Masayoshi; Ichimura, Kazuo; Onozaki, Takashi; Yamaguchi, Hiroyasu; Sasaki, Nobuhiro; Miyahara, Taira; Nishizaki, Yuzo; Ozeki, Yoshihiro; Nakamura, Noriko; Suzuki, Takamasa; Tanaka, Yoshikazu; Sato, Shusei; Shirasawa, Kenta; Isobe, Sachiko; Miyamura, Yoshinori; Watanabe, Akiko; Nakayama, Shinobu; Kishida, Yoshie; Kohara, Mitsuyo; Tabata, Satoshi
2014-01-01
The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. ‘Francesco’ was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568 887 315 bp, consisting of 45 088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16 644 bp and 60 737 bp, respectively, and the longest scaffold was 1 287 144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp. PMID:24344172
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Zhu, Ying; Zhao, Rui; Piehowski, Paul D.
One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-µm-i.d. columns increase signal intensity by >3-fold relative to those using 75-µm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos mass spectrometer significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap), leading tomore » a ~3× increase in peptide identifications and 1.7× increase in identified protein groups for 2 ng tryptic digests of bacterial lysate. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~ 95% for 0.5 ng samples and by ~42% for 2 ng samples. The present platform is capable of identifying >3000 protein groups from tryptic digestion of cell lysates equivalent to 50 HeLa cells and 100 THP-1 cells (~10 ng total proteins), respectively, and >950 proteins from subnanogram bacterial and archaeal cell lysates. The present ultrasensitive LC-MS platform is expected to enable deep proteome coverage for subnanogram samples, including single mammalian cells.« less
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Benson, Dennis A; Karsch-Mizrachi, Ilene; Lipman, David J; Ostell, James; Wheeler, David L
2007-01-01
GenBank (R) is a comprehensive database that contains publicly available nucleotide sequences for more than 240 000 named organisms, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the web-based BankIt or standalone Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the EMBL Data Library in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through NCBI's retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI Homepage (www.ncbi.nlm.nih.gov).
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Benson, Dennis A; Karsch-Mizrachi, Ilene; Lipman, David J; Ostell, James; Wheeler, David L
2005-01-01
GenBank is a comprehensive database that contains publicly available DNA sequences for more than 165,000 named organisms, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the web-based BankIt or standalone Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the EMBL Data Library in the UK and the DNA Data Bank of Japan helps to ensure worldwide coverage. GenBank is accessible through NCBI's retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, go to the NCBI Homepage at http://www.ncbi.nlm.nih.gov.
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Benson, Dennis A; Karsch-Mizrachi, Ilene; Lipman, David J; Ostell, James; Wheeler, David L
2006-01-01
GenBank (R) is a comprehensive database that contains publicly available DNA sequences for more than 205 000 named organisms, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the Web-based BankIt or standalone Sequin programs and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the EMBL Data Library in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through NCBI's retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, go to the NCBI Homepage at www.ncbi.nlm.nih.gov.
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Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Kim, Kwang-Youl; Kwon, Kyung-Hoon; Yoo, Jong Shin; Omenn, Gilbert S; Baker, Mark S; Hancock, William S; Paik, Young-Ki
2015-12-04
Approximately 2.9 billion long base-pair human reference genome sequences are known to encode some 20 000 representative proteins. However, 3000 proteins, that is, ~15% of all proteins, have no or very weak proteomic evidence and are still missing. Missing proteins may be present in rare samples in very low abundance or be only temporarily expressed, causing problems in their detection and protein profiling. In particular, some technical limitations cause missing proteins to remain unassigned. For example, current mass spectrometry techniques have high limits and error rates for the detection of complex biological samples. An insufficient proteome coverage in a reference sequence database and spectral library also raises major issues. Thus, the development of a better strategy that results in greater sensitivity and accuracy in the search for missing proteins is necessary. To this end, we used a new strategy, which combines a reference spectral library search and a simulated spectral library search, to identify missing proteins. We built the human iRefSPL, which contains the original human reference spectral library and additional peptide sequence-spectrum match entries from other species. We also constructed the human simSPL, which contains the simulated spectra of 173 907 human tryptic peptides determined by MassAnalyzer (version 2.3.1). To prove the enhanced analytical performance of the combination of the human iRefSPL and simSPL methods for the identification of missing proteins, we attempted to reanalyze the placental tissue data set (PXD000754). The data from each experiment were analyzed using PeptideProphet, and the results were combined using iProphet. For the quality control, we applied the class-specific false-discovery rate filtering method. All of the results were filtered at a false-discovery rate of <1% at the peptide and protein levels. The quality-controlled results were then cross-checked with the neXtProt DB (2014-09-19 release). The two spectral libraries, iRefSPL and simSPL, were designed to ensure no overlap of the proteome coverage. They were shown to be complementary to spectral library searching and significantly increased the number of matches. From this trial, 12 new missing proteins were identified that passed the following criterion: at least 2 peptides of 7 or more amino acids in length or one of 9 or more amino acids in length with one or more unique sequences. Thus, the iRefSPL and simSPL combination can be used to help identify peptides that have not been detected by conventional sequence database searches with improved sensitivity and a low error rate.
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Expanded microbial genome coverage and improved protein family annotation in the COG database
Galperin, Michael Y.; Makarova, Kira S.; Wolf, Yuri I.; Koonin, Eugene V.
2015-01-01
Microbial genome sequencing projects produce numerous sequences of deduced proteins, only a small fraction of which have been or will ever be studied experimentally. This leaves sequence analysis as the only feasible way to annotate these proteins and assign to them tentative functions. The Clusters of Orthologous Groups of proteins (COGs) database (http://www.ncbi.nlm.nih.gov/COG/), first created in 1997, has been a popular tool for functional annotation. Its success was largely based on (i) its reliance on complete microbial genomes, which allowed reliable assignment of orthologs and paralogs for most genes; (ii) orthology-based approach, which used the function(s) of the characterized member(s) of the protein family (COG) to assign function(s) to the entire set of carefully identified orthologs and describe the range of potential functions when there were more than one; and (iii) careful manual curation of the annotation of the COGs, aimed at detailed prediction of the biological function(s) for each COG while avoiding annotation errors and overprediction. Here we present an update of the COGs, the first since 2003, and a comprehensive revision of the COG annotations and expansion of the genome coverage to include representative complete genomes from all bacterial and archaeal lineages down to the genus level. This re-analysis of the COGs shows that the original COG assignments had an error rate below 0.5% and allows an assessment of the progress in functional genomics in the past 12 years. During this time, functions of many previously uncharacterized COGs have been elucidated and tentative functional assignments of many COGs have been validated, either by targeted experiments or through the use of high-throughput methods. A particularly important development is the assignment of functions to several widespread, conserved proteins many of which turned out to participate in translation, in particular rRNA maturation and tRNA modification. The new version of the COGs is expected to become an important tool for microbial genomics. PMID:25428365
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Tertiary alphabet for the observable protein structural universe.
Mackenzie, Craig O; Zhou, Jianfu; Grigoryan, Gevorg
2016-11-22
Here, we systematically decompose the known protein structural universe into its basic elements, which we dub tertiary structural motifs (TERMs). A TERM is a compact backbone fragment that captures the secondary, tertiary, and quaternary environments around a given residue, comprising one or more disjoint segments (three on average). We seek the set of universal TERMs that capture all structure in the Protein Data Bank (PDB), finding remarkable degeneracy. Only ∼600 TERMs are sufficient to describe 50% of the PDB at sub-Angstrom resolution. However, more rare geometries also exist, and the overall structural coverage grows logarithmically with the number of TERMs. We go on to show that universal TERMs provide an effective mapping between sequence and structure. We demonstrate that TERM-based statistics alone are sufficient to recapitulate close-to-native sequences given either NMR or X-ray backbones. Furthermore, sequence variability predicted from TERM data agrees closely with evolutionary variation. Finally, locations of TERMs in protein chains can be predicted from sequence alone based on sequence signatures emergent from TERM instances in the PDB. For multisegment motifs, this method identifies spatially adjacent fragments that are not contiguous in sequence-a major bottleneck in structure prediction. Although all TERMs recur in diverse proteins, some appear specialized for certain functions, such as interface formation, metal coordination, or even water binding. Structural biology has benefited greatly from previously observed degeneracies in structure. The decomposition of the known structural universe into a finite set of compact TERMs offers exciting opportunities toward better understanding, design, and prediction of protein structure.
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NASA Astrophysics Data System (ADS)
Smargiasso, Nicolas; Quinton, Loic; de Pauw, Edwin
2012-03-01
One of the mechanisms leading to MALDI in-source decay (MALDI ISD) is the transfer of hydrogen radicals to analytes upon laser irradiation. Analytes such as peptides or proteins may undergo ISD and this method can therefore be exploited for top-down sequencing. When performed on peptides, radical-induced ISD results in production of c- and z-ions, as also found in ETD and ECD activation. Here, we describe two new compounds which, when used as MALDI matrices, are able to efficiently induce ISD of peptides and proteins: 2-aminobenzamide and 2-aminobenzoic acid. In-source reduction of the disulfide bridge containing peptide Calcitonin further confirmed the radicalar mechanism of the ISD process. ISD of peptides led, in addition to c- and z-ions, to the generation of a-, x-, and y-ions both in positive and in negative ion modes. Finally, good sequence coverage was obtained for the sequencing of myoglobin (17 kDa protein), confirming the effectiveness of both 2-aminobenzamide and 2-aminobenzoic acid as MALDI ISD matrices.
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Smargiasso, Nicolas; Quinton, Loic; De Pauw, Edwin
2012-03-01
One of the mechanisms leading to MALDI in-source decay (MALDI ISD) is the transfer of hydrogen radicals to analytes upon laser irradiation. Analytes such as peptides or proteins may undergo ISD and this method can therefore be exploited for top-down sequencing. When performed on peptides, radical-induced ISD results in production of c- and z-ions, as also found in ETD and ECD activation. Here, we describe two new compounds which, when used as MALDI matrices, are able to efficiently induce ISD of peptides and proteins: 2-aminobenzamide and 2-aminobenzoic acid. In-source reduction of the disulfide bridge containing peptide Calcitonin further confirmed the radicalar mechanism of the ISD process. ISD of peptides led, in addition to c- and z-ions, to the generation of a-, x-, and y-ions both in positive and in negative ion modes. Finally, good sequence coverage was obtained for the sequencing of myoglobin (17 kDa protein), confirming the effectiveness of both 2-aminobenzamide and 2-aminobenzoic acid as MALDI ISD matrices.
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Bazak, Lily; Haviv, Ami; Barak, Michal; Jacob-Hirsch, Jasmine; Deng, Patricia; Zhang, Rui; Isaacs, Farren J; Rechavi, Gideon; Li, Jin Billy; Eisenberg, Eli; Levanon, Erez Y
2014-03-01
RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity are not yet clear. Here, we analyzed large-scale RNA-seq data and detected ∼1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultradeep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual antisense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.
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Asamizu, Erika; Nakamura, Yasukazu; Sato, Shusei; Tabata, Satoshi
2004-02-01
To perform a comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 74472 3'-end expressed sequence tags (EST) were generated from cDNA libraries produced from six different organs. Clustering of sequences was performed with an identity criterion of 95% for 50 bases, and a total of 20457 non-redundant sequences, 8503 contigs and 11954 singletons were generated. EST sequence coverage was analyzed by using the annotated L. japonicus genomic sequence and 1093 of the 1889 predicted protein-encoding genes (57.9%) were hit by the EST sequence(s). Gene content was compared to several plant species. Among the 8503 contigs, 471 were identified as sequences conserved only in leguminous species and these included several disease resistance-related genes. This suggested that in legumes, these genes may have evolved specifically to resist pathogen attack. The rate of gene sequence divergence was assessed by comparing similarity level and functional category based on the Gene Ontology (GO) annotation of Arabidopsis genes. This revealed that genes encoding ribosomal proteins, as well as those related to translation, photosynthesis, and cellular structure were more abundantly represented in the highly conserved class, and that genes encoding transcription factors and receptor protein kinases were abundantly represented in the less conserved class. To make the sequence information and the cDNA clones available to the research community, a Web database with useful services was created at http://www.kazusa.or.jp/en/plant/lotus/EST/.
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Metagenomic Taxonomy-Guided Database-Searching Strategy for Improving Metaproteomic Analysis.
Xiao, Jinqiu; Tanca, Alessandro; Jia, Ben; Yang, Runqing; Wang, Bo; Zhang, Yu; Li, Jing
2018-04-06
Metaproteomics provides a direct measure of the functional information by investigating all proteins expressed by a microbiota. However, due to the complexity and heterogeneity of microbial communities, it is very hard to construct a sequence database suitable for a metaproteomic study. Using a public database, researchers might not be able to identify proteins from poorly characterized microbial species, while a sequencing-based metagenomic database may not provide adequate coverage for all potentially expressed protein sequences. To address this challenge, we propose a metagenomic taxonomy-guided database-search strategy (MT), in which a merged database is employed, consisting of both taxonomy-guided reference protein sequences from public databases and proteins from metagenome assembly. By applying our MT strategy to a mock microbial mixture, about two times as many peptides were detected as with the metagenomic database only. According to the evaluation of the reliability of taxonomic attribution, the rate of misassignments was comparable to that obtained using an a priori matched database. We also evaluated the MT strategy with a human gut microbial sample, and we found 1.7 times as many peptides as using a standard metagenomic database. In conclusion, our MT strategy allows the construction of databases able to provide high sensitivity and precision in peptide identification in metaproteomic studies, enabling the detection of proteins from poorly characterized species within the microbiota.
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Lee, Imchang; Chalita, Mauricio; Ha, Sung-Min; Na, Seong-In; Yoon, Seok-Hwan; Chun, Jongsik
2017-06-01
Thanks to the recent advancement of DNA sequencing technology, the cost and time of prokaryotic genome sequencing have been dramatically decreased. It has repeatedly been reported that genome sequencing using high-throughput next-generation sequencing is prone to contaminations due to its high depth of sequencing coverage. Although a few bioinformatics tools are available to detect potential contaminations, these have inherited limitations as they only use protein-coding genes. Here we introduce a new algorithm, called ContEst16S, to detect potential contaminations using 16S rRNA genes from genome assemblies. We screened 69 745 prokaryotic genomes from the NCBI Assembly Database using ContEst16S and found that 594 were contaminated by bacteria, human and plants. Of the predicted contaminated genomes, 8 % were not predicted by the existing protein-coding gene-based tool, implying that both methods can be complementary in the detection of contaminations. A web-based service of the algorithm is available at www.ezbiocloud.net/tools/contest16s.
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Benson, Dennis A; Karsch-Mizrachi, Ilene; Lipman, David J; Ostell, James; Sayers, Eric W
2010-01-01
GenBank is a comprehensive database that contains publicly available nucleotide sequences for more than 300,000 organisms named at the genus level or lower, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole genome shotgun (WGS) and environmental sampling projects. Most submissions are made using the web-based BankIt or standalone Sequin programs, and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the European Molecular Biology Laboratory Nucleotide Sequence Database in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through the NCBI Entrez retrieval system, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bi-monthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI homepage: www.ncbi.nlm.nih.gov.
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Benson, Dennis A; Karsch-Mizrachi, Ilene; Lipman, David J; Ostell, James; Sayers, Eric W
2009-01-01
GenBank is a comprehensive database that contains publicly available nucleotide sequences for more than 300,000 organisms named at the genus level or lower, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Most submissions are made using the web-based BankIt or standalone Sequin programs, and accession numbers are assigned by GenBank(R) staff upon receipt. Daily data exchange with the European Molecular Biology Laboratory Nucleotide Sequence Database in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through the National Center for Biotechnology Information (NCBI) Entrez retrieval system, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI Homepage: www.ncbi.nlm.nih.gov.
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Madsen, James A.; Xu, Hua; Robinson, Michelle R.; Horton, Andrew P.; Shaw, Jared B.; Giles, David K.; Kaoud, Tamer S.; Dalby, Kevin N.; Trent, M. Stephen; Brodbelt, Jennifer S.
2013-01-01
The use of ultraviolet photodissociation (UVPD) for the activation and dissociation of peptide anions is evaluated for broader coverage of the proteome. To facilitate interpretation and assignment of the resulting UVPD mass spectra of peptide anions, the MassMatrix database search algorithm was modified to allow automated analysis of negative polarity MS/MS spectra. The new UVPD algorithms were developed based on the MassMatrix database search engine by adding specific fragmentation pathways for UVPD. The new UVPD fragmentation pathways in MassMatrix were rigorously and statistically optimized using two large data sets with high mass accuracy and high mass resolution for both MS1 and MS2 data acquired on an Orbitrap mass spectrometer for complex Halobacterium and HeLa proteome samples. Negative mode UVPD led to the identification of 3663 and 2350 peptides for the Halo and HeLa tryptic digests, respectively, corresponding to 655 and 645 peptides that were unique when compared with electron transfer dissociation (ETD), higher energy collision-induced dissociation, and collision-induced dissociation results for the same digests analyzed in the positive mode. In sum, 805 and 619 proteins were identified via UVPD for the Halobacterium and HeLa samples, respectively, with 49 and 50 unique proteins identified in contrast to the more conventional MS/MS methods. The algorithm also features automated charge determination for low mass accuracy data, precursor filtering (including intact charge-reduced peaks), and the ability to combine both positive and negative MS/MS spectra into a single search, and it is freely open to the public. The accuracy and specificity of the MassMatrix UVPD search algorithm was also assessed for low resolution, low mass accuracy data on a linear ion trap. Analysis of a known mixture of three mitogen-activated kinases yielded similar sequence coverage percentages for UVPD of peptide anions versus conventional collision-induced dissociation of peptide cations, and when these methods were combined into a single search, an increase of up to 13% sequence coverage was observed for the kinases. The ability to sequence peptide anions and cations in alternating scans in the same chromatographic run was also demonstrated. Because ETD has a significant bias toward identifying highly basic peptides, negative UVPD was used to improve the identification of the more acidic peptides in conjunction with positive ETD for the more basic species. In this case, tryptic peptides from the cytosolic section of HeLa cells were analyzed by polarity switching nanoLC-MS/MS utilizing ETD for cation sequencing and UVPD for anion sequencing. Relative to searching using ETD alone, positive/negative polarity switching significantly improved sequence coverages across identified proteins, resulting in a 33% increase in unique peptide identifications and more than twice the number of peptide spectral matches. PMID:23695934
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Sequence analysis of the genome of carnation (Dianthus caryophyllus L.).
Yagi, Masafumi; Kosugi, Shunichi; Hirakawa, Hideki; Ohmiya, Akemi; Tanase, Koji; Harada, Taro; Kishimoto, Kyutaro; Nakayama, Masayoshi; Ichimura, Kazuo; Onozaki, Takashi; Yamaguchi, Hiroyasu; Sasaki, Nobuhiro; Miyahara, Taira; Nishizaki, Yuzo; Ozeki, Yoshihiro; Nakamura, Noriko; Suzuki, Takamasa; Tanaka, Yoshikazu; Sato, Shusei; Shirasawa, Kenta; Isobe, Sachiko; Miyamura, Yoshinori; Watanabe, Akiko; Nakayama, Shinobu; Kishida, Yoshie; Kohara, Mitsuyo; Tabata, Satoshi
2014-06-01
The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. 'Francesco' was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568,887,315 bp, consisting of 45,088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16,644 bp and 60,737 bp, respectively, and the longest scaffold was 1,287,144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼ 98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp. © The Author 2013. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.
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MassSieve: Panning MS/MS peptide data for proteins
Slotta, Douglas J.; McFarland, Melinda A.; Markey, Sanford P.
2010-01-01
We present MassSieve, a Java-based platform for visualization and parsimony analysis of single and comparative LC-MS/MS database search engine results. The success of mass spectrometric peptide sequence assignment algorithms has led to the need for a tool to merge and evaluate the increasing data set sizes that result from LC-MS/MS-based shotgun proteomic experiments. MassSieve supports reports from multiple search engines with differing search characteristics, which can increase peptide sequence coverage and/or identify conflicting or ambiguous spectral assignments. PMID:20564260
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Sequencing of Seven Haloarchaeal Genomes Reveals Patterns of Genomic Flux
Lynch, Erin A.; Langille, Morgan G. I.; Darling, Aaron; Wilbanks, Elizabeth G.; Haltiner, Caitlin; Shao, Katie S. Y.; Starr, Michael O.; Teiling, Clotilde; Harkins, Timothy T.; Edwards, Robert A.; Eisen, Jonathan A.; Facciotti, Marc T.
2012-01-01
We report the sequencing of seven genomes from two haloarchaeal genera, Haloferax and Haloarcula. Ease of cultivation and the existence of well-developed genetic and biochemical tools for several diverse haloarchaeal species make haloarchaea a model group for the study of archaeal biology. The unique physiological properties of these organisms also make them good candidates for novel enzyme discovery for biotechnological applications. Seven genomes were sequenced to ∼20×coverage and assembled to an average of 50 contigs (range 5 scaffolds - 168 contigs). Comparisons of protein-coding gene compliments revealed large-scale differences in COG functional group enrichment between these genera. Analysis of genes encoding machinery for DNA metabolism reveals genera-specific expansions of the general transcription factor TATA binding protein as well as a history of extensive duplication and horizontal transfer of the proliferating cell nuclear antigen. Insights gained from this study emphasize the importance of haloarchaea for investigation of archaeal biology. PMID:22848480
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Efficient encapsulation of proteins with random copolymers.
Nguyen, Trung Dac; Qiao, Baofu; Olvera de la Cruz, Monica
2018-06-12
Membraneless organelles are aggregates of disordered proteins that form spontaneously to promote specific cellular functions in vivo. The possibility of synthesizing membraneless organelles out of cells will therefore enable fabrication of protein-based materials with functions inherent to biological matter. Since random copolymers contain various compositions and sequences of solvophobic and solvophilic groups, they are expected to function in nonbiological media similarly to a set of disordered proteins in membraneless organelles. Interestingly, the internal environment of these organelles has been noted to behave more like an organic solvent than like water. Therefore, an adsorbed layer of random copolymers that mimics the function of disordered proteins could, in principle, protect and enhance the proteins' enzymatic activity even in organic solvents, which are ideal when the products and/or the reactants have limited solubility in aqueous media. Here, we demonstrate via multiscale simulations that random copolymers efficiently incorporate proteins into different solvents with the potential to optimize their enzymatic activity. We investigate the key factors that govern the ability of random copolymers to encapsulate proteins, including the adsorption energy, copolymer average composition, and solvent selectivity. The adsorbed polymer chains have remarkably similar sequences, indicating that the proteins are able to select certain sequences that best reduce their exposure to the solvent. We also find that the protein surface coverage decreases when the fluctuation in the average distance between the protein adsorption sites increases. The results herein set the stage for computational design of random copolymers for stabilizing and delivering proteins across multiple media.
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Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.
Raghav, Sunil Kumar; Deplancke, Bart
2012-01-01
Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.
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Barker, F. Keith; Oyler-McCance, Sara; Tomback, Diana F.
2015-01-01
Next generation sequencing methods allow rapid, economical accumulation of data that have many applications, even at relatively low levels of genome coverage. However, the utility of shotgun sequencing data sets for specific goals may vary depending on the biological nature of the samples sequenced. We show that the ability to assemble mitogenomes from three avian samples of two different tissue types varies widely. In particular, data with coverage typical of microsatellite development efforts (∼1×) from DNA extracted from avian blood failed to cover even 50% of the mitogenome, relative to at least 500-fold coverage from muscle-derived data. Researchers should consider possible applications of their data and select the tissue source for their work accordingly. Practitioners analyzing low-coverage shotgun sequencing data (including for microsatellite locus development) should consider the potential benefits of mitogenome assembly, including internal barcode verification of species identity, mitochondrial primer development, and phylogenetics.
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Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics*
Zhang, Xi
2015-01-01
The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea–trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting—not destroying—structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. PMID:26081834
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Genome Pool Strategy for Structural Coverage of Protein Families
Jaroszewski, Lukasz; Slabinski, Lukasz; Wooley, John; Deacon, Ashley M.; Lesley, Scott A.; Wilson, Ian. A.; Godzik, Adam
2010-01-01
As noticed by generations of structural biologists, closely homologous proteins may have substantially different crystallization properties and propensities. These observations can be used to systematically introduce additional dimensionality into crystallization trials by targeting homologous proteins from multiple genomes in a “genome pool” strategy. Through extensive use of our recently introduced “crystallization feasibility score” (Slabinski et al., 2007a), we can explain that the genome pool strategy works well because the crystallization feasibility scores are surprisingly broad within families of homologous proteins, with most families containing a range of optimal to very difficult targets. We also show that some families can be regarded as relatively “easy”, where a significant number of proteins are predicted to have optimal crystallization features, and others are “very difficult”, where almost none are predicted to result in a crystal structure. Thus, the outcome of such variable distributions of such crystallizability' preferences leads to uneven structural coverage of known families, with “easier” or “optimal” families having several times more solved structures than “very difficult” ones. Nevertheless, this latter category can be successfully targeted by increasing the number of genomes that are used to select targets from a given family. On average, adding 10 new genomes to the “genome pool” provides more promising targets for 7 “very difficult” families. In contrast, our crystallization feasibility score does not indicate that any specific microbial genomes can be readily classified as “easier” or “very difficult” with respect to providing suitable candidates for crystallization and structure determination. Finally, our analyses show that specific physicochemical properties of the protein sequence favor successful outcomes for structure determination and, hence, the group of proteins with known 3D structures is systematically different from the general pool of known proteins. We, therefore, assess the structural consequences of these differences in protein sequence and protein biophysical properties. PMID:19000818
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Tertiary alphabet for the observable protein structural universe
Mackenzie, Craig O.; Zhou, Jianfu; Grigoryan, Gevorg
2016-01-01
Here, we systematically decompose the known protein structural universe into its basic elements, which we dub tertiary structural motifs (TERMs). A TERM is a compact backbone fragment that captures the secondary, tertiary, and quaternary environments around a given residue, comprising one or more disjoint segments (three on average). We seek the set of universal TERMs that capture all structure in the Protein Data Bank (PDB), finding remarkable degeneracy. Only ∼600 TERMs are sufficient to describe 50% of the PDB at sub-Angstrom resolution. However, more rare geometries also exist, and the overall structural coverage grows logarithmically with the number of TERMs. We go on to show that universal TERMs provide an effective mapping between sequence and structure. We demonstrate that TERM-based statistics alone are sufficient to recapitulate close-to-native sequences given either NMR or X-ray backbones. Furthermore, sequence variability predicted from TERM data agrees closely with evolutionary variation. Finally, locations of TERMs in protein chains can be predicted from sequence alone based on sequence signatures emergent from TERM instances in the PDB. For multisegment motifs, this method identifies spatially adjacent fragments that are not contiguous in sequence—a major bottleneck in structure prediction. Although all TERMs recur in diverse proteins, some appear specialized for certain functions, such as interface formation, metal coordination, or even water binding. Structural biology has benefited greatly from previously observed degeneracies in structure. The decomposition of the known structural universe into a finite set of compact TERMs offers exciting opportunities toward better understanding, design, and prediction of protein structure. PMID:27810958
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Wheeler, David
2007-01-01
GenBank(R) is a comprehensive database of publicly available DNA sequences for more than 205,000 named organisms and for more than 60,000 within the embryophyta, obtained through submissions from individual laboratories and batch submissions from large-scale sequencing projects. Daily data exchange with the European Molecular Biology Laboratory (EMBL) in Europe and the DNA Data Bank of Japan ensures worldwide coverage. GenBank is accessible through the National Center for Biotechnology Information (NCBI) retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases with taxonomy, genome, mapping, protein structure, and domain information and the biomedical journal literature through PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available through FTP. GenBank usage scenarios ranging from local analyses of the data available through FTP to online analyses supported by the NCBI Web-based tools are discussed. To access GenBank and its related retrieval and analysis services, go to the NCBI Homepage at http://www.ncbi.nlm.nih.gov.
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Benson, Dennis A; Karsch-Mizrachi, Ilene; Lipman, David J; Ostell, James; Sayers, Eric W
2011-01-01
GenBank® is a comprehensive database that contains publicly available nucleotide sequences for more than 380,000 organisms named at the genus level or lower, obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole genome shotgun (WGS) and environmental sampling projects. Most submissions are made using the web-based BankIt or standalone Sequin programs, and accession numbers are assigned by GenBank staff upon receipt. Daily data exchange with the European Nucleotide Archive (ENA) and the DNA Data Bank of Japan (DDBJ) ensures worldwide coverage. GenBank is accessible through the NCBI Entrez retrieval system that integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of the GenBank database are available by FTP. To access GenBank and its related retrieval and analysis services, begin at the NCBI Homepage: www.ncbi.nlm.nih.gov.
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Network-based function prediction and interactomics: the case for metabolic enzymes.
Janga, S C; Díaz-Mejía, J Javier; Moreno-Hagelsieb, G
2011-01-01
As sequencing technologies increase in power, determining the functions of unknown proteins encoded by the DNA sequences so produced becomes a major challenge. Functional annotation is commonly done on the basis of amino-acid sequence similarity alone. Long after sequence similarity becomes undetectable by pair-wise comparison, profile-based identification of homologs can often succeed due to the conservation of position-specific patterns, important for a protein's three dimensional folding and function. Nevertheless, prediction of protein function from homology-driven approaches is not without problems. Homologous proteins might evolve different functions and the power of homology detection has already started to reach its maximum. Computational methods for inferring protein function, which exploit the context of a protein in cellular networks, have come to be built on top of homology-based approaches. These network-based functional inference techniques provide both a first hand hint into a proteins' functional role and offer complementary insights to traditional methods for understanding the function of uncharacterized proteins. Most recent network-based approaches aim to integrate diverse kinds of functional interactions to boost both coverage and confidence level. These techniques not only promise to solve the moonlighting aspect of proteins by annotating proteins with multiple functions, but also increase our understanding on the interplay between different functional classes in a cell. In this article we review the state of the art in network-based function prediction and describe some of the underlying difficulties and successes. Given the volume of high-throughput data that is being reported the time is ripe to employ these network-based approaches, which can be used to unravel the functions of the uncharacterized proteins accumulating in the genomic databases. © 2010 Elsevier Inc. All rights reserved.
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Schilmiller, Anthony L; Miner, Dennis P; Larson, Matthew; McDowell, Eric; Gang, David R; Wilkerson, Curtis; Last, Robert L
2010-07-01
Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces beta-caryophyllene and alpha-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells.
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Schilmiller, Anthony L.; Miner, Dennis P.; Larson, Matthew; McDowell, Eric; Gang, David R.; Wilkerson, Curtis; Last, Robert L.
2010-01-01
Shotgun proteomics analysis allows hundreds of proteins to be identified and quantified from a single sample at relatively low cost. Extensive DNA sequence information is a prerequisite for shotgun proteomics, and it is ideal to have sequence for the organism being studied rather than from related species or accessions. While this requirement has limited the set of organisms that are candidates for this approach, next generation sequencing technologies make it feasible to obtain deep DNA sequence coverage from any organism. As part of our studies of specialized (secondary) metabolism in tomato (Solanum lycopersicum) trichomes, 454 sequencing of cDNA was combined with shotgun proteomics analyses to obtain in-depth profiles of genes and proteins expressed in leaf and stem glandular trichomes of 3-week-old plants. The expressed sequence tag and proteomics data sets combined with metabolite analysis led to the discovery and characterization of a sesquiterpene synthase that produces β-caryophyllene and α-humulene from E,E-farnesyl diphosphate in trichomes of leaf but not of stem. This analysis demonstrates the utility of combining high-throughput cDNA sequencing with proteomics experiments in a target tissue. These data can be used for dissection of other biochemical processes in these specialized epidermal cells. PMID:20431087
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Ruggles, Kelly V; Tang, Zuojian; Wang, Xuya; Grover, Himanshu; Askenazi, Manor; Teubl, Jennifer; Cao, Song; McLellan, Michael D; Clauser, Karl R; Tabb, David L; Mertins, Philipp; Slebos, Robbert; Erdmann-Gilmore, Petra; Li, Shunqiang; Gunawardena, Harsha P; Xie, Ling; Liu, Tao; Zhou, Jian-Ying; Sun, Shisheng; Hoadley, Katherine A; Perou, Charles M; Chen, Xian; Davies, Sherri R; Maher, Christopher A; Kinsinger, Christopher R; Rodland, Karen D; Zhang, Hui; Zhang, Zhen; Ding, Li; Townsend, R Reid; Rodriguez, Henry; Chan, Daniel; Smith, Richard D; Liebler, Daniel C; Carr, Steven A; Payne, Samuel; Ellis, Matthew J; Fenyő, David
2016-03-01
Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations, and splice variants identified in cancer cells are translated. Herein, we apply a proteogenomic data integration tool (QUILTS) to illustrate protein variant discovery using whole genome, whole transcriptome, and global proteome datasets generated from a pair of luminal and basal-like breast-cancer-patient-derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS sample process replicates defined here as an independent tandem MS experiment using identical sample material. Despite analysis of over 30 sample process replicates, only about 10% of SNVs (somatic and germline) detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNVs without a detectable mRNA transcript were also observed, suggesting that transcriptome coverage was incomplete (∼80%). In contrast to germline variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than in the luminal tumor, raising the possibility of differential translation or protein degradation effects. In conclusion, this large-scale proteogenomic integration allowed us to determine the degree to which mutations are translated and identify gaps in sequence coverage, thereby benchmarking current technology and progress toward whole cancer proteome and transcriptome analysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Ramirez-Sarmiento, Cesar A; Komives, Elizabeth A
2018-04-06
Hydrogen-deuterium exchange mass spectrometry (HDXMS) has emerged as a powerful approach for revealing folding and allostery in protein-protein interactions. The advent of higher resolution mass spectrometers combined with ion mobility separation and ultra performance liquid chromatographic separations have allowed the complete coverage of large protein sequences and multi-protein complexes. Liquid-handling robots have improved the reproducibility and accurate temperature control of the sample preparation. Many researchers are also appreciating the power of combining biophysical approaches such as stopped-flow fluorescence, single molecule FRET, and molecular dynamics simulations with HDXMS. In this review, we focus on studies that have used a combination of approaches to reveal (re)folding of proteins as well as on long-distance allosteric changes upon interaction. Copyright © 2018 Elsevier Inc. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ruggles, Kelly V.; Tang, Zuojian; Wang, Xuya
Improvements in mass spectrometry (MS)-based peptide sequencing provide a new opportunity to determine whether polymorphisms, mutations and splice variants identified in cancer cells are translated. Herein we therefore describe a proteogenomic data integration tool (QUILTS) and illustrate its application to whole genome, transcriptome and global MS peptide sequence datasets generated from a pair of luminal and basal-like breast cancer patient derived xenografts (PDX). The sensitivity of proteogenomic analysis for singe nucleotide variant (SNV) expression and novel splice junction (NSJ) detection was probed using multiple MS/MS process replicates. Despite over thirty sample replicates, only about 10% of all SNV (somatic andmore » germline) were detected by both DNA and RNA sequencing were observed as peptides. An even smaller proportion of peptides corresponding to NSJ observed by RNA sequencing were detected (<0.1%). Peptides mapping to DNA-detected SNV without a detectable mRNA transcript were also observed demonstrating the transcriptome coverage was also incomplete (~80%). In contrast to germ-line variants, somatic variants were less likely to be detected at the peptide level in the basal-like tumor than the luminal tumor raising the possibility of differential translation or protein degradation effects. In conclusion, the QUILTS program integrates DNA, RNA and peptide sequencing to assess the degree to which somatic mutations are translated and therefore biologically active. By identifying gaps in sequence coverage QUILTS benchmarks current technology and assesses progress towards whole cancer proteome and transcriptome analysis.« less
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Walia, Rasna R; Xue, Li C; Wilkins, Katherine; El-Manzalawy, Yasser; Dobbs, Drena; Honavar, Vasant
2014-01-01
Protein-RNA interactions are central to essential cellular processes such as protein synthesis and regulation of gene expression and play roles in human infectious and genetic diseases. Reliable identification of protein-RNA interfaces is critical for understanding the structural bases and functional implications of such interactions and for developing effective approaches to rational drug design. Sequence-based computational methods offer a viable, cost-effective way to identify putative RNA-binding residues in RNA-binding proteins. Here we report two novel approaches: (i) HomPRIP, a sequence homology-based method for predicting RNA-binding sites in proteins; (ii) RNABindRPlus, a new method that combines predictions from HomPRIP with those from an optimized Support Vector Machine (SVM) classifier trained on a benchmark dataset of 198 RNA-binding proteins. Although highly reliable, HomPRIP cannot make predictions for the unaligned parts of query proteins and its coverage is limited by the availability of close sequence homologs of the query protein with experimentally determined RNA-binding sites. RNABindRPlus overcomes these limitations. We compared the performance of HomPRIP and RNABindRPlus with that of several state-of-the-art predictors on two test sets, RB44 and RB111. On a subset of proteins for which homologs with experimentally determined interfaces could be reliably identified, HomPRIP outperformed all other methods achieving an MCC of 0.63 on RB44 and 0.83 on RB111. RNABindRPlus was able to predict RNA-binding residues of all proteins in both test sets, achieving an MCC of 0.55 and 0.37, respectively, and outperforming all other methods, including those that make use of structure-derived features of proteins. More importantly, RNABindRPlus outperforms all other methods for any choice of tradeoff between precision and recall. An important advantage of both HomPRIP and RNABindRPlus is that they rely on readily available sequence and sequence-derived features of RNA-binding proteins. A webserver implementation of both methods is freely available at http://einstein.cs.iastate.edu/RNABindRPlus/.
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Mazandu, Gaston K; Mulder, Nicola J
2012-07-01
Despite ever-increasing amounts of sequence and functional genomics data, there is still a deficiency of functional annotation for many newly sequenced proteins. For Mycobacterium tuberculosis (MTB), more than half of its genome is still uncharacterized, which hampers the search for new drug targets within the bacterial pathogen and limits our understanding of its pathogenicity. As for many other genomes, the annotations of proteins in the MTB proteome were generally inferred from sequence homology, which is effective but its applicability has limitations. We have carried out large-scale biological data integration to produce an MTB protein functional interaction network. Protein functional relationships were extracted from the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, and additional functional interactions from microarray, sequence and protein signature data. The confidence level of protein relationships in the additional functional interaction data was evaluated using a dynamic data-driven scoring system. This functional network has been used to predict functions of uncharacterized proteins using Gene Ontology (GO) terms, and the semantic similarity between these terms measured using a state-of-the-art GO similarity metric. To achieve better trade-off between improvement of quality, genomic coverage and scalability, this prediction is done by observing the key principles driving the biological organization of the functional network. This study yields a new functionally characterized MTB strain CDC1551 proteome, consisting of 3804 and 3698 proteins out of 4195 with annotations in terms of the biological process and molecular function ontologies, respectively. These data can contribute to research into the Development of effective anti-tubercular drugs with novel biological mechanisms of action. Copyright © 2011 Elsevier B.V. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Mushegian, Arcady R., E-mail: mushegian2@gmail.com; Elena, Santiago F., E-mail: sfelena@ibmcp.upv.es; The Santa Fe Institute, Santa Fe, NM 87501
Homologs of Tobacco mosaic virus 30K cell-to-cell movement protein are encoded by diverse plant viruses. Mechanisms of action and evolutionary origins of these proteins remain obscure. We expand the picture of conservation and evolution of the 30K proteins, producing sequence alignment of the 30K superfamily with the broadest phylogenetic coverage thus far and illuminating structural features of the core all-beta fold of these proteins. Integrated copies of pararetrovirus 30K movement genes are prevalent in euphyllophytes, with at least one copy intact in nearly every examined species, and mRNAs detected for most of them. Sequence analysis suggests repeated integrations, pseudogenizations, andmore » positive selection in those provirus genes. An unannotated 30K-superfamily gene in Arabidopsis thaliana genome is likely expressed as a fusion with the At1g37113 transcript. This molecular background of endopararetrovirus gene products in plants may change our view of virus infection and pathogenesis, and perhaps of cellular homeostasis in the hosts. - Highlights: • Sequence region shared by plant virus “30K” movement proteins has an all-beta fold. • Most euphyllophyte genomes contain integrated copies of pararetroviruses. • These integrated virus genomes often include intact movement protein genes. • Molecular evidence suggests that these “30K” genes may be selected for function.« less
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ACLAME: a CLAssification of Mobile genetic Elements, update 2010.
Leplae, Raphaël; Lima-Mendez, Gipsi; Toussaint, Ariane
2010-01-01
The ACLAME database is dedicated to the collection, analysis and classification of sequenced mobile genetic elements (MGEs, in particular phages and plasmids). In addition to providing information on the MGEs content, classifications are available at various levels of organization. At the gene/protein level, families group similar sequences that are expected to share the same function. Families of four or more proteins are manually assigned with a functional annotation using the GeneOntology and the locally developed ontology MeGO dedicated to MGEs. At the genome level, evolutionary cohesive modules group sets of protein families shared among MGEs. At the population level, networks display the reticulate evolutionary relationships among MGEs. To increase the coverage of the phage sequence space, ACLAME version 0.4 incorporates 760 high-quality predicted prophages selected from the Prophinder database. Most of the data can be downloaded from the freely accessible ACLAME web site (http://aclame.ulb.ac.be). The BLAST interface for querying the database has been extended and numerous tools for in-depth analysis of the results have been added.
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Variant calling in low-coverage whole genome sequencing of a Native American population sample.
Bizon, Chris; Spiegel, Michael; Chasse, Scott A; Gizer, Ian R; Li, Yun; Malc, Ewa P; Mieczkowski, Piotr A; Sailsbery, Josh K; Wang, Xiaoshu; Ehlers, Cindy L; Wilhelmsen, Kirk C
2014-01-30
The reduction in the cost of sequencing a human genome has led to the use of genotype sampling strategies in order to impute and infer the presence of sequence variants that can then be tested for associations with traits of interest. Low-coverage Whole Genome Sequencing (WGS) is a sampling strategy that overcomes some of the deficiencies seen in fixed content SNP array studies. Linkage-disequilibrium (LD) aware variant callers, such as the program Thunder, may provide a calling rate and accuracy that makes a low-coverage sequencing strategy viable. We examined the performance of an LD-aware variant calling strategy in a population of 708 low-coverage whole genome sequences from a community sample of Native Americans. We assessed variant calling through a comparison of the sequencing results to genotypes measured in 641 of the same subjects using a fixed content first generation exome array. The comparison was made using the variant calling routines GATK Unified Genotyper program and the LD-aware variant caller Thunder. Thunder was found to improve concordance in a coverage dependent fashion, while correctly calling nearly all of the common variants as well as a high percentage of the rare variants present in the sample. Low-coverage WGS is a strategy that appears to collect genetic information intermediate in scope between fixed content genotyping arrays and deep-coverage WGS. Our data suggests that low-coverage WGS is a viable strategy with a greater chance of discovering novel variants and associations than fixed content arrays for large sample association analyses.
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Genome sequence of Phytophthora ramorum: implications for management
Brett Tyler; Sucheta Tripathy; Nik Grunwald; Kurt Lamour; Kelly Ivors; Matteo Garbelotto; Daniel Rokhsar; Nik Putnam; Igor Grigoriev; Jeffrey Boore
2006-01-01
A draft genome sequence has been determined for Phytophthora ramorum, together with a draft sequence of the soybean pathogen Phytophthora sojae. The P. ramorum genome was sequenced to a depth of 7-fold coverage, while the P. sojae genome was sequenced to a depth of 9-fold coverage. The genome...
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Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia
Carninci, Piero; Waki, Kazunori; Shiraki, Toshiyuki; Konno, Hideaki; Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Arakawa, Takahiro; Ishii, Yoshiyuki; Sasaki, Daisuke; Bono, Hidemasa; Kondo, Shinji; Sugahara, Yuichi; Saito, Rintaro; Osato, Naoki; Fukuda, Shiro; Sato, Kenjiro; Watahiki, Akira; Hirozane-Kishikawa, Tomoko; Nakamura, Mari; Shibata, Yuko; Yasunishi, Ayako; Kikuchi, Noriko; Yoshiki, Atsushi; Kusakabe, Moriaki; Gustincich, Stefano; Beisel, Kirk; Pavan, William; Aidinis, Vassilis; Nakagawara, Akira; Held, William A.; Iwata, Hiroo; Kono, Tomohiro; Nakauchi, Hiromitsu; Lyons, Paul; Wells, Christine; Hume, David A.; Fagiolini, Michela; Hensch, Takao K.; Brinkmeier, Michelle; Camper, Sally; Hirota, Junji; Mombaerts, Peter; Muramatsu, Masami; Okazaki, Yasushi; Kawai, Jun; Hayashizaki, Yoshihide
2003-01-01
We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3′-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5′ end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5′-end clusters identify regions that are potential promoters for 8637 known genes and 5′-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete. PMID:12819125
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Zhu, Xun; Xie, Shangbo; Armengaud, Jean; Xie, Wen; Guo, Zhaojiang; Kang, Shi; Wu, Qingjun; Wang, Shaoli; Xia, Jixing; He, Rongjun; Zhang, Youjun
2016-01-01
The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept that has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multialgorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a data set comprising 2694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. To get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a nonmodel animal. As the first tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest. PMID:26902207
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Expanded microbial genome coverage and improved protein family annotation in the COG database.
Galperin, Michael Y; Makarova, Kira S; Wolf, Yuri I; Koonin, Eugene V
2015-01-01
Microbial genome sequencing projects produce numerous sequences of deduced proteins, only a small fraction of which have been or will ever be studied experimentally. This leaves sequence analysis as the only feasible way to annotate these proteins and assign to them tentative functions. The Clusters of Orthologous Groups of proteins (COGs) database (http://www.ncbi.nlm.nih.gov/COG/), first created in 1997, has been a popular tool for functional annotation. Its success was largely based on (i) its reliance on complete microbial genomes, which allowed reliable assignment of orthologs and paralogs for most genes; (ii) orthology-based approach, which used the function(s) of the characterized member(s) of the protein family (COG) to assign function(s) to the entire set of carefully identified orthologs and describe the range of potential functions when there were more than one; and (iii) careful manual curation of the annotation of the COGs, aimed at detailed prediction of the biological function(s) for each COG while avoiding annotation errors and overprediction. Here we present an update of the COGs, the first since 2003, and a comprehensive revision of the COG annotations and expansion of the genome coverage to include representative complete genomes from all bacterial and archaeal lineages down to the genus level. This re-analysis of the COGs shows that the original COG assignments had an error rate below 0.5% and allows an assessment of the progress in functional genomics in the past 12 years. During this time, functions of many previously uncharacterized COGs have been elucidated and tentative functional assignments of many COGs have been validated, either by targeted experiments or through the use of high-throughput methods. A particularly important development is the assignment of functions to several widespread, conserved proteins many of which turned out to participate in translation, in particular rRNA maturation and tRNA modification. The new version of the COGs is expected to become an important tool for microbial genomics. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by US Government employees and is in the public domain in the US.
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Top-down Mass Spectrometry of Cardiac Myofilament Proteins in Health and Disease
Ying, Peng; Serife, Ayaz-Guner; Deyang, Yu; Ying, Ge
2014-01-01
Myofilaments are composed of thin and thick filaments which coordinate with each other to regulate muscle contraction and relaxation. Posttranslational modifications (PTMs) together with genetic variations and alternative splicing of the myofilament proteins play essential roles in regulating cardiac contractility in health and disease. Therefore, a comprehensive characterization of the myofilament proteins in physiological and pathological conditions is essential for better understanding the molecular basis of cardiac function and dysfunction. Due to the vast complexity and dynamic nature of proteins, it is challenging to obtain a holistic view of myofilament protein modifications. In recent years, top-down mass spectrometry (MS) has emerged as a powerful approach to study isoform composition and PTMs of proteins owing to its advantage of complete sequence coverage and its ability to identify PTMs and sequence variants without a priori knowledge. In this review, we will discuss the application of top-down MS to study cardiac myofilaments and highlight the insights it provides into the understanding of molecular mechanisms in contractile dysfunction of heart failure. Particularly, recent results of cardiac troponin and tropomyosin modifications will be elaborated. The limitations and perspectives on the use of top-down MS for myofilament protein characterization will also be briefly discussed. PMID:24945106
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Functionalizing Microporous Membranes for Protein Purification and Protein Digestion
NASA Astrophysics Data System (ADS)
Dong, Jinlan; Bruening, Merlin L.
2015-07-01
This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.
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Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.
Dong, Jinlan; Bruening, Merlin L
2015-01-01
This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.
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Less is More: Membrane Protein Digestion Beyond Urea-Trypsin Solution for Next-level Proteomics.
Zhang, Xi
2015-09-01
The goal of next-level bottom-up membrane proteomics is protein function investigation, via high-coverage high-throughput peptide-centric quantitation of expression, modifications and dynamic structures at systems scale. Yet efficient digestion of mammalian membrane proteins presents a daunting barrier, and prevalent day-long urea-trypsin in-solution digestion proved insufficient to reach this goal. Many efforts contributed incremental advances over past years, but involved protein denaturation that disconnected measurement from functional states. Beyond denaturation, the recent discovery of structure/proteomics omni-compatible detergent n-dodecyl-β-d-maltopyranoside, combined with pepsin and PNGase F columns, enabled breakthroughs in membrane protein digestion: a 2010 DDM-low-TCEP (DLT) method for H/D-exchange (HDX) using human G protein-coupled receptor, and a 2015 flow/detergent-facilitated protease and de-PTM digestions (FDD) for integrative deep sequencing and quantitation using full-length human ion channel complex. Distinguishing protein solubilization from denaturation, protease digestion reliability from theoretical specificity, and reduction from alkylation, these methods shifted day(s)-long paradigms into minutes, and afforded fully automatable (HDX)-protein-peptide-(tandem mass tag)-HPLC pipelines to instantly measure functional proteins at deep coverage, high peptide reproducibility, low artifacts and minimal leakage. Promoting-not destroying-structures and activities harnessed membrane proteins for the next-level streamlined functional proteomics. This review analyzes recent advances in membrane protein digestion methods and highlights critical discoveries for future proteomics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Zhu, Ying; Zhao, Rui; Piehowski, Paul D.; ...
2017-09-01
One of the greatest challenges for mass spectrometry (MS)-based proteomics is the limited ability to analyze small samples. Here in this study, we investigate the relative contributions of liquid chromatography (LC), MS instrumentation and data analysis methods with the aim of improving proteome coverage for sample sizes ranging from 0.5 ng to 50 ng. We show that the LC separations utilizing 30-μm-i.d. columns increase signal intensity by >3-fold relative to those using 75-μm-i.d. columns, leading to 32% increase in peptide identifications. The Orbitrap Fusion Lumos MS significantly boosted both sensitivity and sequencing speed relative to earlier generation Orbitraps (e.g., LTQ-Orbitrap),more » leading to a ~3-fold increase in peptide identifications and 1.7-fold increase in identified protein groups for 2 ng tryptic digests of the bacterium S. oneidensis. The Match Between Runs algorithm of open-source MaxQuant software further increased proteome coverage by ~95% for 0.5 ng samples and by ~42% for 2 ng samples. Using the best combination of the above variables, we were able to identify >3,000 proteins from 10 ng tryptic digests from both HeLa and THP-1 mammalian cell lines. We also identified >950 proteins from subnanogram archaeal/bacterial cocultures. Finally, the present ultrasensitive LC-MS platform achieves a level of proteome coverage not previously realized for ultra-small sample loadings, and is expected to facilitate the analysis of subnanogram samples, including single mammalian cells.« less
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Luo, Chengwei; Tsementzi, Despina; Kyrpides, Nikos; Read, Timothy; Konstantinidis, Konstantinos T
2012-01-01
Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R(2)>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.
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A high-coverage draft genome of the mycalesine butterfly Bicyclus anynana.
Nowell, Reuben W; Elsworth, Ben; Oostra, Vicencio; Zwaan, Bas J; Wheat, Christopher W; Saastamoinen, Marjo; Saccheri, Ilik J; Van't Hof, Arjen E; Wasik, Bethany R; Connahs, Heidi; Aslam, Muhammad L; Kumar, Sujai; Challis, Richard J; Monteiro, Antónia; Brakefield, Paul M; Blaxter, Mark
2017-07-01
The mycalesine butterfly Bicyclus anynana, the "Squinting bush brown," is a model organism in the study of lepidopteran ecology, development, and evolution. Here, we present a draft genome sequence for B. anynana to serve as a genomics resource for current and future studies of this important model species. Seven libraries with insert sizes ranging from 350 bp to 20 kb were constructed using DNA from an inbred female and sequenced using both Illumina and PacBio technology; 128 Gb of raw Illumina data was filtered to 124 Gb and assembled to a final size of 475 Mb (∼×260 assembly coverage). Contigs were scaffolded using mate-pair, transcriptome, and PacBio data into 10 800 sequences with an N50 of 638 kb (longest scaffold 5 Mb). The genome is comprised of 26% repetitive elements and encodes a total of 22 642 predicted protein-coding genes. Recovery of a BUSCO set of core metazoan genes was almost complete (98%). Overall, these metrics compare well with other recently published lepidopteran genomes. We report a high-quality draft genome sequence for Bicyclus anynana. The genome assembly and annotated gene models are available at LepBase (http://ensembl.lepbase.org/index.html). © The Authors 2017. Published by Oxford University Press.
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A high-coverage draft genome of the mycalesine butterfly Bicyclus anynana
Elsworth, Ben; Oostra, Vicencio; Zwaan, Bas J.; Wheat, Christopher W.; Saastamoinen, Marjo; Saccheri, Ilik J.; van’t Hof, Arjen E.; Wasik, Bethany R.; Connahs, Heidi; Aslam, Muhammad L.; Kumar, Sujai; Challis, Richard J.; Monteiro, Antónia; Brakefield, Paul M.
2017-01-01
Abstract The mycalesine butterfly Bicyclus anynana, the “Squinting bush brown,” is a model organism in the study of lepidopteran ecology, development, and evolution. Here, we present a draft genome sequence for B. anynana to serve as a genomics resource for current and future studies of this important model species. Seven libraries with insert sizes ranging from 350 bp to 20 kb were constructed using DNA from an inbred female and sequenced using both Illumina and PacBio technology; 128 Gb of raw Illumina data was filtered to 124 Gb and assembled to a final size of 475 Mb (∼×260 assembly coverage). Contigs were scaffolded using mate-pair, transcriptome, and PacBio data into 10 800 sequences with an N50 of 638 kb (longest scaffold 5 Mb). The genome is comprised of 26% repetitive elements and encodes a total of 22 642 predicted protein-coding genes. Recovery of a BUSCO set of core metazoan genes was almost complete (98%). Overall, these metrics compare well with other recently published lepidopteran genomes. We report a high-quality draft genome sequence for Bicyclus anynana. The genome assembly and annotated gene models are available at LepBase (http://ensembl.lepbase.org/index.html). PMID:28486658
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Tetteh, Kevin K A; Conway, David J
2011-10-13
Merozoite surface protein 1 (MSP1) of Plasmodium falciparum has been implicated as an important target of acquired immunity, and candidate components for a vaccine include polymorphic epitopes in the N-terminal polymorphic block 2 region. We designed a polyvalent hybrid recombinant protein incorporating sequences of the three major allelic types of block 2 together with a composite repeat sequence of one of the types and N-terminal flanking T cell epitopes, and compared this with a series of recombinant proteins containing modular sub-components and similarly expressed in Escherichia coli. Immunogenicity of the full polyvalent hybrid protein was tested in both mice and rabbits, and comparative immunogenicity studies of the sub-component modules were performed in mice. The full hybrid protein induced high titre antibodies against each of the major block 2 allelic types expressed as separate recombinant proteins and against a wide range of allelic types naturally expressed by a panel of diverse P. falciparum isolates, while the sub-component modules had partial antigenic coverage as expected. This encourages further development and evaluation of the full MSP1 block 2 polyvalent hybrid protein as a candidate blood-stage component of a malaria vaccine. Copyright © 2011 Elsevier Ltd. All rights reserved.
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Szabó, Zsolt; Gyula, Péter; Robotka, Hermina; Bató, Emese; Gálik, Bence; Pach, Péter; Pekker, Péter; Papp, Ildikó; Bihari, Zoltán
2015-01-01
Methylibium sp. strain T29 was isolated from a gasoline-contaminated aquifer and proved to have excellent capabilities in degrading some common fuel oxygenates like methyl tert-butyl ether, tert-amyl methyl ether and tert-butyl alcohol along with other organic compounds. Here, we report the draft genome sequence of M. sp. strain T29 together with the description of the genome properties and its annotation. The draft genome consists of 608 contigs with a total size of 4,449,424 bp and an average coverage of 150×. The genome exhibits an average G + C content of 68.7 %, and contains 4754 protein coding and 52 RNA genes, including 48 tRNA genes. 71 % of the protein coding genes could be assigned to COG (Clusters of Orthologous Groups) categories. A formerly unknown circular plasmid designated as pT29A was isolated and sequenced separately and found to be 86,856 bp long.
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Chen, Wenzhang; Shen, Jing; Yin, Xuefeng; Yu, Yingnian
2007-01-01
A nano-scale solid-phase extraction (SPE) device was developed for the detection of gel-separated proteins in low abundance by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) with a simplified microfabrication technology. By using SU-8 photoresist instead of epoxy glue to connect the microchannel and transfer capillary, polymeric contaminant signals in MS analysis were significantly reduced. Micro SPE columns with different capacities and geometric characteristics were investigated in order to increase the detection sensitivity and decrease spot size for MALDI-TOF-MS analysis. It is shown that enhancements in sensitivities for the detection of proteins in low abundance were correlated with the reduction in column capacity and increase in column aspect ratio. Fifty nanoliters of matrix solution were sufficient to elute the sample completely from the optimized micro SPE column with 3.5 nL capacity. The mass spectrum of a 5 fmol in-gel tryptic digest of bovine serum albumin (BSA), processed by the micro SPE column, demonstrated that 29 peptides matched the protein giving a sequence coverage of 51%, which was better than that obtained from analysis of 25 fmol of the same sample prepared by the dried-droplet method. With the micro SPE column treatment of 2 microL of digestion supernatant of a gel spot of the IQGAP1 protein, 15 peptides were detected from the mass spectrum with the highest individual score of 111, while, with a ZipTip procedure, only nine peaks were detected with the highest individual score of 71. Analytical results demonstrated that this approach greatly improved the sequence coverage and identification specificity for the tested protein. It can serve as a very useful tool in proteomics studies, especially for low abundance proteins. Copyright (c) 2006 John Wiley & Sons, Ltd.
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Thompson, Kirsten F; Patel, Selina; Williams, Liam; Tsai, Peter; Constantine, Rochelle; Baker, C Scott; Millar, Craig D
2016-01-01
Using an Illumina platform, we shot-gun sequenced the complete mitochondrial genome of Gray's beaked whale (Mesoplodon grayi) to an average coverage of 152X. We performed a de novo assembly using SOAPdenovo2 and determined the total mitogenome length to be 16,347 bp. The nucleotide composition was asymmetric (33.3% A, 24.6% C, 12.6% G, 29.5% T) with an overall GC content of 37.2%. The gene organization was similar to that of other cetaceans with 13 protein-coding genes, 2 rRNAs (12S and 16S), 22 predicted tRNAs and 1 control region or D-loop. We found no evidence of heteroplasmy or nuclear copies of mitochondrial DNA in this individual. Beaked whales within the genus Mesoplodon are rarely seen at sea and their basic biology is poorly understood. These data will contribute to resolving the phylogeography and population ecology of this speciose group.
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An Annotated Draft Genome for Radix auricularia (Gastropoda, Mollusca)
Feldmeyer, Barbara; Schmidt, Hanno; Greshake, Bastian; Tills, Oliver; Truebano, Manuela; Rundle, Simon D.; Paule, Juraj; Ebersberger, Ingo; Pfenninger, Markus
2017-01-01
Molluscs are the second most species-rich phylum in the animal kingdom, yet only 11 genomes of this group have been published so far. Here, we present the draft genome sequence of the pulmonate freshwater snail Radix auricularia. Six whole genome shotgun libraries with different layouts were sequenced. The resulting assembly comprises 4,823 scaffolds with a cumulative length of 910 Mb and an overall read coverage of 72×. The assembly contains 94.6% of a metazoan core gene collection, indicating an almost complete coverage of the coding fraction. The discrepancy of ∼690 Mb compared with the estimated genome size of R. auricularia (1.6 Gb) results from a high repeat content of 70% mainly comprising DNA transposons. The annotation of 17,338 protein coding genes was supported by the use of publicly available transcriptome data. This draft will serve as starting point for further genomic and population genetic research in this scientifically important phylum. PMID:28204581
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USDA-ARS?s Scientific Manuscript database
Next-generation sequencing technology such as genotyping-by-sequencing (GBS) made low-cost, but often low-coverage, whole-genome sequencing widely available. Extensive inbreeding in crop plants provides an untapped, high quality source of phased haplotypes for imputing missing genotypes. We introduc...
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The Reference Genome of the Halophytic Plant Eutrema salsugineum
Yang, Ruolin; Jarvis, David E.; Chen, Hao; Beilstein, Mark A.; Grimwood, Jane; Jenkins, Jerry; Shu, ShengQiang; Prochnik, Simon; Xin, Mingming; Ma, Chuang; Schmutz, Jeremy; Wing, Rod A.; Mitchell-Olds, Thomas; Schumaker, Karen S.; Wang, Xiangfeng
2013-01-01
Halophytes are plants that can naturally tolerate high concentrations of salt in the soil, and their tolerance to salt stress may occur through various evolutionary and molecular mechanisms. Eutrema salsugineum is a halophytic species in the Brassicaceae that can naturally tolerate multiple types of abiotic stresses that typically limit crop productivity, including extreme salinity and cold. It has been widely used as a laboratorial model for stress biology research in plants. Here, we present the reference genome sequence (241 Mb) of E. salsugineum at 8× coverage sequenced using the traditional Sanger sequencing-based approach with comparison to its close relative Arabidopsis thaliana. The E. salsugineum genome contains 26,531 protein-coding genes and 51.4% of its genome is composed of repetitive sequences that mostly reside in pericentromeric regions. Comparative analyses of the genome structures, protein-coding genes, microRNAs, stress-related pathways, and estimated translation efficiency of proteins between E. salsugineum and A. thaliana suggest that halophyte adaptation to environmental stresses may occur via a global network adjustment of multiple regulatory mechanisms. The E. salsugineum genome provides a resource to identify naturally occurring genetic alterations contributing to the adaptation of halophytic plants to salinity and that might be bioengineered in related crop species. PMID:23518688
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Modeling genome coverage in single-cell sequencing
Daley, Timothy; Smith, Andrew D.
2014-01-01
Motivation: Single-cell DNA sequencing is necessary for examining genetic variation at the cellular level, which remains hidden in bulk sequencing experiments. But because they begin with such small amounts of starting material, the amount of information that is obtained from single-cell sequencing experiment is highly sensitive to the choice of protocol employed and variability in library preparation. In particular, the fraction of the genome represented in single-cell sequencing libraries exhibits extreme variability due to quantitative biases in amplification and loss of genetic material. Results: We propose a method to predict the genome coverage of a deep sequencing experiment using information from an initial shallow sequencing experiment mapped to a reference genome. The observed coverage statistics are used in a non-parametric empirical Bayes Poisson model to estimate the gain in coverage from deeper sequencing. This approach allows researchers to know statistical features of deep sequencing experiments without actually sequencing deeply, providing a basis for optimizing and comparing single-cell sequencing protocols or screening libraries. Availability and implementation: The method is available as part of the preseq software package. Source code is available at http://smithlabresearch.org/preseq. Contact: andrewds@usc.edu Supplementary information: Supplementary material is available at Bioinformatics online. PMID:25107873
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ChIP-seq: advantages and challenges of a maturing technology.
Park, Peter J
2009-10-01
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes. Owing to the tremendous progress in next-generation sequencing technology, ChIP-seq offers higher resolution, less noise and greater coverage than its array-based predecessor ChIP-chip. With the decreasing cost of sequencing, ChIP-seq has become an indispensable tool for studying gene regulation and epigenetic mechanisms. In this Review, I describe the benefits and challenges in harnessing this technique with an emphasis on issues related to experimental design and data analysis. ChIP-seq experiments generate large quantities of data, and effective computational analysis will be crucial for uncovering biological mechanisms.
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BIPS: BIANA Interolog Prediction Server. A tool for protein-protein interaction inference.
Garcia-Garcia, Javier; Schleker, Sylvia; Klein-Seetharaman, Judith; Oliva, Baldo
2012-07-01
Protein-protein interactions (PPIs) play a crucial role in biology, and high-throughput experiments have greatly increased the coverage of known interactions. Still, identification of complete inter- and intraspecies interactomes is far from being complete. Experimental data can be complemented by the prediction of PPIs within an organism or between two organisms based on the known interactions of the orthologous genes of other organisms (interologs). Here, we present the BIANA (Biologic Interactions and Network Analysis) Interolog Prediction Server (BIPS), which offers a web-based interface to facilitate PPI predictions based on interolog information. BIPS benefits from the capabilities of the framework BIANA to integrate the several PPI-related databases. Additional metadata can be used to improve the reliability of the predicted interactions. Sensitivity and specificity of the server have been calculated using known PPIs from different interactomes using a leave-one-out approach. The specificity is between 72 and 98%, whereas sensitivity varies between 1 and 59%, depending on the sequence identity cut-off used to calculate similarities between sequences. BIPS is freely accessible at http://sbi.imim.es/BIPS.php.
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Optimal de novo design of MRM experiments for rapid assay development in targeted proteomics.
Bertsch, Andreas; Jung, Stephan; Zerck, Alexandra; Pfeifer, Nico; Nahnsen, Sven; Henneges, Carsten; Nordheim, Alfred; Kohlbacher, Oliver
2010-05-07
Targeted proteomic approaches such as multiple reaction monitoring (MRM) overcome problems associated with classical shotgun mass spectrometry experiments. Developing MRM quantitation assays can be time consuming, because relevant peptide representatives of the proteins must be found and their retention time and the product ions must be determined. Given the transitions, hundreds to thousands of them can be scheduled into one experiment run. However, it is difficult to select which of the transitions should be included into a measurement. We present a novel algorithm that allows the construction of MRM assays from the sequence of the targeted proteins alone. This enables the rapid development of targeted MRM experiments without large libraries of transitions or peptide spectra. The approach relies on combinatorial optimization in combination with machine learning techniques to predict proteotypicity, retention time, and fragmentation of peptides. The resulting potential transitions are scheduled optimally by solving an integer linear program. We demonstrate that fully automated construction of MRM experiments from protein sequences alone is possible and over 80% coverage of the targeted proteins can be achieved without further optimization of the assay.
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Markoutsa, Stavroula; Bahr, Ute; Papasotiriou, Dimitrios G; Häfner, Ann-Kathrin; Karas, Michael; Sorg, Bernd L
2014-03-01
The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo-NHS-SS-biotin derivatization of lysine side chain can help to detect PTMs in lysine-rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5-lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Falkner, Jayson; Andrews, Philip
2005-05-15
Comparing tandem mass spectra (MSMS) against a known dataset of protein sequences is a common method for identifying unknown proteins; however, the processing of MSMS by current software often limits certain applications, including comprehensive coverage of post-translational modifications, non-specific searches and real-time searches to allow result-dependent instrument control. This problem deserves attention as new mass spectrometers provide the ability for higher throughput and as known protein datasets rapidly grow in size. New software algorithms need to be devised in order to address the performance issues of conventional MSMS protein dataset-based protein identification. This paper describes a novel algorithm based on converting a collection of monoisotopic, centroided spectra to a new data structure, named 'peptide finite state machine' (PFSM), which may be used to rapidly search a known dataset of protein sequences, regardless of the number of spectra searched or the number of potential modifications examined. The algorithm is verified using a set of commercially available tryptic digest protein standards analyzed using an ABI 4700 MALDI TOFTOF mass spectrometer, and a free, open source PFSM implementation. It is illustrated that a PFSM can accurately search large collections of spectra against large datasets of protein sequences (e.g. NCBI nr) using a regular desktop PC; however, this paper only details the method for identifying peptide and subsequently protein candidates from a dataset of known protein sequences. The concept of using a PFSM as a peptide pre-screening technique for MSMS-based search engines is validated by using PFSM with Mascot and XTandem. Complete source code, documentation and examples for the reference PFSM implementation are freely available at the Proteome Commons, http://www.proteomecommons.org and source code may be used both commercially and non-commercially as long as the original authors are credited for their work.
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Anderson, Tavis K; Laegreid, William W; Cerutti, Francesco; Osorio, Fernando A; Nelson, Eric A; Christopher-Hennings, Jane; Goldberg, Tony L
2012-06-15
The extraordinary genetic and antigenic variability of RNA viruses is arguably the greatest challenge to the development of broadly effective vaccines. No single viral variant can induce sufficiently broad immunity, and incorporating all known naturally circulating variants into one multivalent vaccine is not feasible. Furthermore, no objective strategies currently exist to select actual viral variants that should be included or excluded in polyvalent vaccines. To address this problem, we demonstrate a method based on graph theory that quantifies the relative importance of viral variants. We demonstrate our method through application to the envelope glycoprotein gene of a particularly diverse RNA virus of pigs: porcine reproductive and respiratory syndrome virus (PRRSV). Using distance matrices derived from sequence nucleotide difference, amino acid difference and evolutionary distance, we constructed viral networks and used common network statistics to assign each sequence an objective ranking of relative 'importance'. To validate our approach, we use an independent published algorithm to score our top-ranked wild-type variants for coverage of putative T-cell epitopes across the 9383 sequences in our dataset. Top-ranked viruses achieve significantly higher coverage than low-ranked viruses, and top-ranked viruses achieve nearly equal coverage as a synthetic mosaic protein constructed in silico from the same set of 9383 sequences. Our approach relies on the network structure of PRRSV but applies to any diverse RNA virus because it identifies subsets of viral variants that are most important to overall viral diversity. We suggest that this method, through the objective quantification of variant importance, provides criteria for choosing viral variants for further characterization, diagnostics, surveillance and ultimately polyvalent vaccine development.
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Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki
2017-12-01
Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.
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Schroeter, Elena R; DeHart, Caroline J; Cleland, Timothy P; Zheng, Wenxia; Thomas, Paul M; Kelleher, Neil L; Bern, Marshall; Schweitzer, Mary H
2017-02-03
Sequence data from biomolecules such as DNA and proteins, which provide critical information for evolutionary studies, have been assumed to be forever outside the reach of dinosaur paleontology. Proteins, which are predicted to have greater longevity than DNA, have been recovered from two nonavian dinosaurs, but these results remain controversial. For proteomic data derived from extinct Mesozoic organisms to reach their greatest potential for investigating questions of phylogeny and paleobiology, it must be shown that peptide sequences can be reliably and reproducibly obtained from fossils and that fragmentary sequences for ancient proteins can be increasingly expanded. To test the hypothesis that peptides can be repeatedly detected and validated from fossil tissues many millions of years old, we applied updated extraction methodology, high-resolution mass spectrometry, and bioinformatics analyses on a Brachylophosaurus canadensis specimen (MOR 2598) from which collagen I peptides were recovered in 2009. We recovered eight peptide sequences of collagen I: two identical to peptides recovered in 2009 and six new peptides. Phylogenetic analyses place the recovered sequences within basal archosauria. When only the new sequences are considered, B. canadensis is grouped more closely to crocodylians, but when all sequences (current and those reported in 2009) are analyzed, B. canadensis is placed more closely to basal birds. The data robustly support the hypothesis of an endogenous origin for these peptides, confirm the idea that peptides can survive in specimens tens of millions of years old, and bolster the validity of the 2009 study. Furthermore, the new data expand the coverage of B. canadensis collagen I (a 33.6% increase in collagen I alpha 1 and 116.7% in alpha 2). Finally, this study demonstrates the importance of reexamining previously studied specimens with updated methods and instrumentation, as we obtained roughly the same amount of sequence data as the previous study with substantially less sample material. Data are available via ProteomeXchange with identifier PXD005087.
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A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.
Agrotis, Alexander; Ketteler, Robin
2015-01-01
CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.
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Huang, Jiqing; Kast, Juergen
2015-08-07
Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.
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The Bologna Annotation Resource (BAR 3.0): improving protein functional annotation
Casadio, Rita
2017-01-01
Abstract BAR 3.0 updates our server BAR (Bologna Annotation Resource) for predicting protein structural and functional features from sequence. We increase data volume, query capabilities and information conveyed to the user. The core of BAR 3.0 is a graph-based clustering procedure of UniProtKB sequences, following strict pairwise similarity criteria (sequence identity ≥40% with alignment coverage ≥90%). Each cluster contains the available annotation downloaded from UniProtKB, GO, PFAM and PDB. After statistical validation, GO terms and PFAM domains are cluster-specific and annotate new sequences entering the cluster after satisfying similarity constraints. BAR 3.0 includes 28 869 663 sequences in 1 361 773 clusters, of which 22.2% (22 241 661 sequences) and 47.4% (24 555 055 sequences) have at least one validated GO term and one PFAM domain, respectively. 1.4% of the clusters (36% of all sequences) include PDB structures and the cluster is associated to a hidden Markov model that allows building template-target alignment suitable for structural modeling. Some other 3 399 026 sequences are singletons. BAR 3.0 offers an improved search interface, allowing queries by UniProtKB-accession, Fasta sequence, GO-term, PFAM-domain, organism, PDB and ligand/s. When evaluated on the CAFA2 targets, BAR 3.0 largely outperforms our previous version and scores among state-of-the-art methods. BAR 3.0 is publicly available and accessible at http://bar.biocomp.unibo.it/bar3. PMID:28453653
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Molecular and immunological characterisation of the glycosylated orange allergen Cit s 1
Pöltl, Gerald; Ahrazem, Oussama; Paschinger, Katharina; Ibañez, M. Dolores; Salcedo, Gabriel; Wilson, Iain B. H.
2010-01-01
The IgE of sera from patients with a history of allergy to oranges (Citrus sinensis) bind a number of proteins in orange extract, including Cit s 1, a germin-like protein. In the present study, we have analysed its immunological cross-reactivity and its molecular nature. Sera from many of the patients examined recognise a range of glycoproteins and neoglycoconjugates containing β1,2-xylose and core α1,3-fucose on their N-glycans. These reagents also inhibited the interaction of Cit s 1 with patients’ sera, thus underlining the critical role of glycosylation in the recognition of this protein by patients’ IgE and extending previous data showing that deglycosylated Cit s 1 does not possess IgE epitopes. In parallel, we examined the peptide sequence and glycan structure of Cit s 1 using mass spectrometric techniques. Indeed, we achieved complete sequence coverage of the mature protein as compared to the translation of an expressed sequence tag cDNA clone and demonstrated that the single N-glycosylation site of this protein carries oligosaccharides with xylose and fucose residues. Due to the presumed requirement for multivalency for in vivo allergenicity, our molecular data showing that Cit s 1 is monovalent as regards glycosylation and that the single N-glycan is the target of the IgE response to this protein, therefore, explain the immunological cross-reactive properties of Cit s 1 as well as its equivocal nature as a clinically-relevant allergen. PMID:17095532
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Warnke, Stephan; Baldauf, Carsten; Bowers, Michael T; Pagel, Kevin; von Helden, Gert
2014-07-23
Ultraviolet photodissociation (UVPD) of gas-phase proteins has attracted increased attention in recent years. This growing interest is largely based on the fact that, in contrast to slow heating techniques such as collision induced dissociation (CID), the cleavage propensity after absorption of UV light is distributed over the entire protein sequence, which can lead to a very high sequence coverage as required in typical top-down proteomics applications. However, in the gas phase, proteins can adopt a multitude of distinct and sometimes coexisting conformations, and it is not clear how this three-dimensional structure affects the UVPD fragmentation behavior. Using ion mobility-UVPD-mass spectrometry in conjunction with molecular dynamics simulations, we provide the first experimental evidence that UVPD is sensitive to the higher order structure of gas-phase proteins. Distinct UVPD spectra were obtained for different extended conformations of 11(+) ubiquitin ions. Assignment of the fragments showed that the majority of differences arise from cis/trans isomerization of one particular proline peptide bond. Seen from a broader perspective, these data highlight the potential of UVPD to be used for the structural analysis of proteins in the gas phase.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Denef, Vincent; Shah, Manesh B; Verberkmoes, Nathan C
The recent surge in microbial genomic sequencing, combined with the development of high-throughput liquid chromatography-mass-spectrometry-based (LC/LC-MS/MS) proteomics, has raised the question of the extent to which genomic information of one strain or environmental sample can be used to profile proteomes of related strains or samples. Even with decreasing sequencing costs, it remains impractical to obtain genomic sequence for every strain or sample analyzed. Here, we evaluate how shotgun proteomics is affected by amino acid divergence between the sample and the genomic database using a probability-based model and a random mutation simulation model constrained by experimental data. To assess the effectsmore » of nonrandom distribution of mutations, we also evaluated identification levels using in silico peptide data from sequenced isolates with average amino acid identities (AAI) varying between 76 and 98%. We compared the predictions to experimental protein identification levels for a sample that was evaluated using a database that included genomic information for the dominant organism and for a closely related variant (95% AAI). The range of models set the boundaries at which half of the proteins in a proteomic experiment can be identified to be 77-92% AAI between orthologs in the sample and database. Consistent with this prediction, experimental data indicated loss of half the identifiable proteins at 90% AAI. Additional analysis indicated a 6.4% reduction of the initial protein coverage per 1% amino acid divergence and total identification loss at 86% AAI. Consequently, shotgun proteomics is capable of cross-strain identifications but avoids most crossspecies false positives.« less
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Prakash, Celine; Haeseler, Arndt Von
2017-03-01
RNA sequencing (RNA-seq) has emerged as the method of choice for measuring the expression of RNAs in a given cell population. In most RNA-seq technologies, sequencing the full length of RNA molecules requires fragmentation into smaller pieces. Unfortunately, the issue of nonuniform sequencing coverage across a genomic feature has been a concern in RNA-seq and is attributed to biases for certain fragments in RNA-seq library preparation and sequencing. To investigate the expected coverage obtained from fragmentation, we develop a simple fragmentation model that is independent of bias from the experimental method and is not specific to the transcript sequence. Essentially, we enumerate all configurations for maximal placement of a given fragment length, F, on transcript length, T, to represent every possible fragmentation pattern, from which we compute the expected coverage profile across a transcript. We extend this model to incorporate general empirical attributes such as read length, fragment length distribution, and number of molecules of the transcript. We further introduce the fragment starting-point, fragment coverage, and read coverage profiles. We find that the expected profiles are not uniform and that factors such as fragment length to transcript length ratio, read length to fragment length ratio, fragment length distribution, and number of molecules influence the variability of coverage across a transcript. Finally, we explore a potential application of the model where, with simulations, we show that it is possible to correctly estimate the transcript copy number for any transcript in the RNA-seq experiment.
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Haeseler, Arndt Von
2017-01-01
Abstract RNA sequencing (RNA-seq) has emerged as the method of choice for measuring the expression of RNAs in a given cell population. In most RNA-seq technologies, sequencing the full length of RNA molecules requires fragmentation into smaller pieces. Unfortunately, the issue of nonuniform sequencing coverage across a genomic feature has been a concern in RNA-seq and is attributed to biases for certain fragments in RNA-seq library preparation and sequencing. To investigate the expected coverage obtained from fragmentation, we develop a simple fragmentation model that is independent of bias from the experimental method and is not specific to the transcript sequence. Essentially, we enumerate all configurations for maximal placement of a given fragment length, F, on transcript length, T, to represent every possible fragmentation pattern, from which we compute the expected coverage profile across a transcript. We extend this model to incorporate general empirical attributes such as read length, fragment length distribution, and number of molecules of the transcript. We further introduce the fragment starting-point, fragment coverage, and read coverage profiles. We find that the expected profiles are not uniform and that factors such as fragment length to transcript length ratio, read length to fragment length ratio, fragment length distribution, and number of molecules influence the variability of coverage across a transcript. Finally, we explore a potential application of the model where, with simulations, we show that it is possible to correctly estimate the transcript copy number for any transcript in the RNA-seq experiment. PMID:27661099
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PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.
Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred
2018-01-01
The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.
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Bielecka, Magdalena K.; Devos, Nathalie; Gilbert, Mélanie; Hung, Miao-Chiu; Weynants, Vincent; Heckels, John E.
2014-01-01
A recombinant macrophage infectivity potentiator (rMIP) protein of Neisseria meningitidis induces significant serum bactericidal antibody production in mice and is a candidate meningococcal vaccine antigen. However, bioinformatics analysis of MIP showed some amino acid sequence similarity to human FK506-binding proteins (FKBPs) in residues 166 to 252 located in the globular domain of the protein. To circumvent the potential concern over generating antibodies that could recognize human proteins, we immunized mice with recombinant truncated type I rMIP proteins that lacked the globular domain and the signal leader peptide (LP) signal sequence (amino acids 1 to 22) and contained the His purification tag at either the N or C terminus (C-term). The immunogenicity of truncated rMIP proteins was compared to that of full (i.e., full-length) rMIP proteins (containing the globular domain) with either an N- or C-terminal His tag and with or without the LP sequence. By comparing the functional murine antibody responses to these various constructs, we determined that C-term His truncated rMIP (−LP) delivered in liposomes induced high levels of antibodies that bound to the surface of wild-type but not Δmip mutant meningococci and showed bactericidal activity against homologous type I MIP (median titers of 128 to 256) and heterologous type II and III (median titers of 256 to 512) strains, thereby providing at least 82% serogroup B strain coverage. In contrast, in constructs lacking the LP, placement of the His tag at the N terminus appeared to abrogate bactericidal activity. The strategy used in this study would obviate any potential concerns regarding the use of MIP antigens for inclusion in bacterial vaccines. PMID:25452551
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Hao, Piliang; Ren, Yan; Dutta, Bamaprasad; Sze, Siu Kwan
2013-04-26
ERLIC and high-pH RP (Hp-RP) have been reported to be promising alternatives to strong cation exchange (SCX) in proteome fractionation. Here we compared the performance of ERLIC, concatenated ERLIC and concatenated Hp-RP in proteome profiling. The protein identification is comparable in these three strategies, but significantly more unique peptides are identified by the two concatenation methods, resulting in a significant increase of the average protein sequence coverage. The pooling of fractions from spaced intervals results in more uniform distribution of peptides in each fraction compared with the chromatogram-based pooling of adjacent fractions. ERLIC fractionates peptides according to their pI and GRAVY values. These properties remains but becomes less remarkable in concatenated ERLIC. In contrast, the average pI and GRAVY values of the peptides are comparable in each fraction in concatenated Hp-RP. ERLIC performs the best in identifying peptides with pI>9 among the three strategies, while concatenated Hp-RP is good at identifying peptides with pI<4. These advantages are useful when either basic or acidic peptides/proteins are analytical targets. The power of ERLIC in identification of basic peptides seems to be due to their efficient separation from acidic peptides. This study facilitates the choice of proper fractionation strategies based on specific objectives. For in-depth proteomic analysis of a cell, tissue and plasma, multidimensional liquid chromatography (MDLC) is still necessary to reduce sample complexity for improving analytical dynamic range and proteome coverage. This work conducts a direct comparison of three promising first-dimensional proteome fractionation methods. They are comparable in identifying proteins, but concatenated ERLIC and concatenated Hp-RP identify significantly more unique peptides than ERLIC. ERLIC is good at analyzing basic peptides, while concatenated Hp-RP performs the best in analyzing acidic peptides with pI<4. This will facilitate the choice of the proper peptide fractionation strategy based on a specific need. A combination of different fractionation strategies can be used to increase the sequence coverage and number of protein identification due to the complementary effect between different methods. Copyright © 2013 Elsevier B.V. All rights reserved.
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Utilization of sequence on relatives to improve analysis of individuals' low-coverage NGS data
USDA-ARS?s Scientific Manuscript database
Low-coverage sequence data is expected to have low call rates under the prevailing paradigm that genotypes are first “called” from sequence data of each individual independently and subsequent analyses (including determination of haplotypes) are dependent on those called genotypes. However, provide...
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The COG database: an updated version includes eukaryotes
Tatusov, Roman L; Fedorova, Natalie D; Jackson, John D; Jacobs, Aviva R; Kiryutin, Boris; Koonin, Eugene V; Krylov, Dmitri M; Mazumder, Raja; Mekhedov, Sergei L; Nikolskaya, Anastasia N; Rao, B Sridhar; Smirnov, Sergei; Sverdlov, Alexander V; Vasudevan, Sona; Wolf, Yuri I; Yin, Jodie J; Natale, Darren A
2003-01-01
Background The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies. Results We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted) proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens), one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (~1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes. Conclusion The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies. PMID:12969510
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Protein interface classification by evolutionary analysis
2012-01-01
Background Distinguishing biologically relevant interfaces from lattice contacts in protein crystals is a fundamental problem in structural biology. Despite efforts towards the computational prediction of interface character, many issues are still unresolved. Results We present here a protein-protein interface classifier that relies on evolutionary data to detect the biological character of interfaces. The classifier uses a simple geometric measure, number of core residues, and two evolutionary indicators based on the sequence entropy of homolog sequences. Both aim at detecting differential selection pressure between interface core and rim or rest of surface. The core residues, defined as fully buried residues (>95% burial), appear to be fundamental determinants of biological interfaces: their number is in itself a powerful discriminator of interface character and together with the evolutionary measures it is able to clearly distinguish evolved biological contacts from crystal ones. We demonstrate that this definition of core residues leads to distinctively better results than earlier definitions from the literature. The stringent selection and quality filtering of structural and sequence data was key to the success of the method. Most importantly we demonstrate that a more conservative selection of homolog sequences - with relatively high sequence identities to the query - is able to produce a clearer signal than previous attempts. Conclusions An evolutionary approach like the one presented here is key to the advancement of the field, which so far was missing an effective method exploiting the evolutionary character of protein interfaces. Its coverage and performance will only improve over time thanks to the incessant growth of sequence databases. Currently our method reaches an accuracy of 89% in classifying interfaces of the Ponstingl 2003 datasets and it lends itself to a variety of useful applications in structural biology and bioinformatics. We made the corresponding software implementation available to the community as an easy-to-use graphical web interface at http://www.eppic-web.org. PMID:23259833
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Zhu, Xun; Xie, Shangbo; Armengaud, Jean; Xie, Wen; Guo, Zhaojiang; Kang, Shi; Wu, Qingjun; Wang, Shaoli; Xia, Jixing; He, Rongjun; Zhang, Youjun
2016-06-01
The diamondback moth, Plutella xylostella (L.), is the major cosmopolitan pest of brassica and other cruciferous crops. Its larval midgut is a dynamic tissue that interfaces with a wide variety of toxicological and physiological processes. The draft sequence of the P. xylostella genome was recently released, but its annotation remains challenging because of the low sequence coverage of this branch of life and the poor description of exon/intron splicing rules for these insects. Peptide sequencing by computational assignment of tandem mass spectra to genome sequence information provides an experimental independent approach for confirming or refuting protein predictions, a concept that has been termed proteogenomics. In this study, we carried out an in-depth proteogenomic analysis to complement genome annotation of P. xylostella larval midgut based on shotgun HPLC-ESI-MS/MS data by means of a multialgorithm pipeline. A total of 876,341 tandem mass spectra were searched against the predicted P. xylostella protein sequences and a whole-genome six-frame translation database. Based on a data set comprising 2694 novel genome search specific peptides, we discovered 439 novel protein-coding genes and corrected 128 existing gene models. To get the most accurate data to seed further insect genome annotation, more than half of the novel protein-coding genes, i.e. 235 over 439, were further validated after RT-PCR amplification and sequencing of the corresponding transcripts. Furthermore, we validated 53 novel alternative splicings. Finally, a total of 6764 proteins were identified, resulting in one of the most comprehensive proteogenomic study of a nonmodel animal. As the first tissue-specific proteogenomics analysis of P. xylostella, this study provides the fundamental basis for high-throughput proteomics and functional genomics approaches aimed at deciphering the molecular mechanisms of resistance and controlling this pest. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Improved hybrid de novo genome assembly of domesticated apple (Malus x domestica).
Li, Xuewei; Kui, Ling; Zhang, Jing; Xie, Yinpeng; Wang, Liping; Yan, Yan; Wang, Na; Xu, Jidi; Li, Cuiying; Wang, Wen; van Nocker, Steve; Dong, Yang; Ma, Fengwang; Guan, Qingmei
2016-08-08
Domesticated apple (Malus × domestica Borkh) is a popular temperate fruit with high nutrient levels and diverse flavors. In 2012, global apple production accounted for at least one tenth of all harvested fruits. A high-quality apple genome assembly is crucial for the selection and breeding of new cultivars. Currently, a single reference genome is available for apple, assembled from 16.9 × genome coverage short reads via Sanger and 454 sequencing technologies. Although a useful resource, this assembly covers only ~89 % of the non-repetitive portion of the genome, and has a relatively short (16.7 kb) contig N50 length. These downsides make it difficult to apply this reference in transcriptive or whole-genome re-sequencing analyses. Here we present an improved hybrid de novo genomic assembly of apple (Golden Delicious), which was obtained from 76 Gb (~102 × genome coverage) Illumina HiSeq data and 21.7 Gb (~29 × genome coverage) PacBio data. The final draft genome is approximately 632.4 Mb, representing ~ 90 % of the estimated genome. The contig N50 size is 111,619 bp, representing a 7 fold improvement. Further annotation analyses predicted 53,922 protein-coding genes and 2,765 non-coding RNA genes. The new apple genome assembly will serve as a valuable resource for investigating complex apple traits at the genomic level. It is not only suitable for genome editing and gene cloning, but also for RNA-seq and whole-genome re-sequencing studies.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Jacobs, Jon M.; Yang, Xiaohua; Luft, Benjamin J.
2005-04-01
The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B. burgdorferi B31 strain and two other strains (ND40, and JD-1) having different Borrelia pathotypes using strong cation exchange fractionation of proteolytic peptides followed by high-resolution, reversed phase capillary liquid chromatography coupled with ion trap tandem mass spectrometric (LC-MS/MS) analysis. Protein identification was facilitated by the availability of the complete B31more » genome sequence. A total of 665 Borrelia proteins were identified representing ~38 % coverage of the theoretical B31 proteome. A significant overlap was observed between the identified proteins in direct comparisons between any two strains (>72%), but distinct differences were observed among identified hypothetical and outer membrane proteins of the three strains. Such a concurrent proteomic overview of three Borrelia strains based upon only the B31 genome sequence is shown to provide significant insights into the presence or absence of specific proteins and a broad overall comparison among strains.« less
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Hart, Elizabeth A; Caccamo, Mario; Harrow, Jennifer L; Humphray, Sean J; Gilbert, James GR; Trevanion, Steve; Hubbard, Tim; Rogers, Jane; Rothschild, Max F
2007-01-01
Background We describe here the sequencing, annotation and comparative analysis of an 8 Mb region of pig chromosome 17, which provides a useful test region to assess coverage and quality for the pig genome sequencing project. We report our findings comparing the annotation of draft sequence assembled at different depths of coverage. Results Within this region we annotated 71 loci, of which 53 are orthologous to human known coding genes. When compared to the syntenic regions in human (20q13.13-q13.33) and mouse (chromosome 2, 167.5 Mb-178.3 Mb), this region was found to be highly conserved with respect to gene order. The most notable difference between the three species is the presence of a large expansion of zinc finger coding genes and pseudogenes on mouse chromosome 2 between Edn3 and Phactr3 that is absent from pig and human. All of our annotation has been made publicly available in the Vertebrate Genome Annotation browser, VEGA. We assessed the impact of coverage on sequence assembly across this region and found, as expected, that increased sequence depth resulted in fewer, longer contigs. One-third of our annotated loci could not be fully re-aligned back to the low coverage version of the sequence, principally because the transcripts are fragmented over several contigs. Conclusion We have demonstrated the considerable advantages of sequencing at increased read depths and discuss the implications that lower coverage sequence may have on subsequent comparative and functional studies, particularly those involving complex loci such as GNAS. PMID:17705864
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Chen, Tsute; Siddiqui, Huma; Olsen, Ingar
2017-01-01
Currently, genome sequences of a total of 19 Porphyromonas gingivalis strains are available, including eight completed genomes (strains W83, ATCC 33277, TDC60, HG66, A7436, AJW4, 381, and A7A1-28) and 11 high-coverage draft sequences (JCVI SC001, F0185, F0566, F0568, F0569, F0570, SJD2, W4087, W50, Ando, and MP4-504) that are assembled into fewer than 300 contigs. The objective was to compare these genomes at both nucleotide and protein sequence levels in order to understand their phylogenetic and functional relatedness. Four copies of 16S rRNA gene sequences were identified in each of the eight complete genomes and one in the other 11 unfinished genomes. These 43 16S rRNA sequences represent only 24 unique sequences and the derived phylogenetic tree suggests a possible evolutionary history for these strains. Phylogenomic comparison based on shared proteins and whole genome nucleotide sequences consistently showed two groups with closely related members: one consisted of ATCC 33277, 381, and HG66, another of W83, W50, and A7436. At least 1,037 core/shared proteins were identified in the 19 P. gingivalis genomes based on the most stringent detecting parameters. Comparative functional genomics based on genome-wide comparisons between NCBI and RAST annotations, as well as additional approaches, revealed functions that are unique or missing in individual P. gingivalis strains, or species-specific in all P. gingivalis strains, when compared to a neighboring species P. asaccharolytica . All the comparative results of this study are available online for download at ftp://www.homd.org/publication_data/20160425/.
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Chen, Tsute; Siddiqui, Huma; Olsen, Ingar
2017-01-01
Currently, genome sequences of a total of 19 Porphyromonas gingivalis strains are available, including eight completed genomes (strains W83, ATCC 33277, TDC60, HG66, A7436, AJW4, 381, and A7A1-28) and 11 high-coverage draft sequences (JCVI SC001, F0185, F0566, F0568, F0569, F0570, SJD2, W4087, W50, Ando, and MP4-504) that are assembled into fewer than 300 contigs. The objective was to compare these genomes at both nucleotide and protein sequence levels in order to understand their phylogenetic and functional relatedness. Four copies of 16S rRNA gene sequences were identified in each of the eight complete genomes and one in the other 11 unfinished genomes. These 43 16S rRNA sequences represent only 24 unique sequences and the derived phylogenetic tree suggests a possible evolutionary history for these strains. Phylogenomic comparison based on shared proteins and whole genome nucleotide sequences consistently showed two groups with closely related members: one consisted of ATCC 33277, 381, and HG66, another of W83, W50, and A7436. At least 1,037 core/shared proteins were identified in the 19 P. gingivalis genomes based on the most stringent detecting parameters. Comparative functional genomics based on genome-wide comparisons between NCBI and RAST annotations, as well as additional approaches, revealed functions that are unique or missing in individual P. gingivalis strains, or species-specific in all P. gingivalis strains, when compared to a neighboring species P. asaccharolytica. All the comparative results of this study are available online for download at ftp://www.homd.org/publication_data/20160425/. PMID:28261563
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Quail, Michael A; Smith, Miriam; Coupland, Paul; Otto, Thomas D; Harris, Simon R; Connor, Thomas R; Bertoni, Anna; Swerdlow, Harold P; Gu, Yong
2012-07-24
Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent's PGM, Pacific Biosciences' RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.
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Multi-Objective Optimization of Spacecraft Trajectories for Small-Body Coverage Missions
NASA Technical Reports Server (NTRS)
Hinckley, David, Jr.; Englander, Jacob; Hitt, Darren
2017-01-01
Visual coverage of surface elements of a small-body object requires multiple images to be taken that meet many requirements on their viewing angles, illumination angles, times of day, and combinations thereof. Designing trajectories capable of maximizing total possible coverage may not be useful since the image target sequence and the feasibility of said sequence given the rotation-rate limitations of the spacecraft are not taken into account. This work presents a means of optimizing, in a multi-objective manner, surface target sequences that account for such limitations.
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NASA Astrophysics Data System (ADS)
Halim, Mohammad A.; MacAleese, Luke; Lemoine, Jérôme; Antoine, Rodolphe; Dugourd, Philippe; Girod, Marion
2018-02-01
Mass spectrometry-based methods have made significant progress in characterizing post-translational modifications in peptides and proteins; however, certain aspects regarding fragmentation methods must still be improved. A good technique is expected to provide excellent sequence information, locate PTM sites, and retain the labile PTM groups. To address these issues, we investigate 10.6 μm IRMPD, 213 nm UVPD, and combined UV and IR photodissociation, known as HiLoPD (high-low photodissociation), for phospho-, sulfo-, and glyco-peptide cations. IRMPD shows excellent backbone fragmentation and produces equal numbers of N- and C-terminal ions. The results reveal that 213 nm UVPD and HiLoPD methods can provide diverse backbone fragmentation producing a/x, b/y, and c/z ions with excellent sequence coverage, locate PTM sites, and offer reasonable retention efficiency for phospho- and glyco-peptides. Excellent sequence coverage is achieved for sulfo-peptides and the position of the SO3 group can be pinpointed; however, widespread SO3 losses are detected irrespective of the methods used herein. Based on the overall performance achieved, we believe that 213 nm UVPD and HiLoPD can serve as alternative options to collision activation and electron transfer dissociations for phospho- and glyco-proteomics.
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FunGene: the functional gene pipeline and repository.
Fish, Jordan A; Chai, Benli; Wang, Qiong; Sun, Yanni; Brown, C Titus; Tiedje, James M; Cole, James R
2013-01-01
Ribosomal RNA genes have become the standard molecular markers for microbial community analysis for good reasons, including universal occurrence in cellular organisms, availability of large databases, and ease of rRNA gene region amplification and analysis. As markers, however, rRNA genes have some significant limitations. The rRNA genes are often present in multiple copies, unlike most protein-coding genes. The slow rate of change in rRNA genes means that multiple species sometimes share identical 16S rRNA gene sequences, while many more species share identical sequences in the short 16S rRNA regions commonly analyzed. In addition, the genes involved in many important processes are not distributed in a phylogenetically coherent manner, potentially due to gene loss or horizontal gene transfer. While rRNA genes remain the most commonly used markers, key genes in ecologically important pathways, e.g., those involved in carbon and nitrogen cycling, can provide important insights into community composition and function not obtainable through rRNA analysis. However, working with ecofunctional gene data requires some tools beyond those required for rRNA analysis. To address this, our Functional Gene Pipeline and Repository (FunGene; http://fungene.cme.msu.edu/) offers databases of many common ecofunctional genes and proteins, as well as integrated tools that allow researchers to browse these collections and choose subsets for further analysis, build phylogenetic trees, test primers and probes for coverage, and download aligned sequences. Additional FunGene tools are specialized to process coding gene amplicon data. For example, FrameBot produces frameshift-corrected protein and DNA sequences from raw reads while finding the most closely related protein reference sequence. These tools can help provide better insight into microbial communities by directly studying key genes involved in important ecological processes.
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Bypassing bacterial infection in phage display by sequencing DNA released from phage particles.
Villequey, Camille; Kong, Xu-Dong; Heinis, Christian
2017-11-01
Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type. A potential strategy for bypassing the bacterial infection step is to directly sequence DNA extracted from phage particles after a single round of phage panning using high-throughput sequencing. In this work, we have quantified the fraction of phage clones that can be identified by directly sequencing DNA from phage particles. The results show that the DNA of essentially all of the phage particles can be 'decoded', and that the sequence coverage for mutants equals that of amplified DNA extracted from cells infected with wild-type phage. This procedure is particularly attractive for selections with phage that have a compromised infection capacity, and it may allow phage display to be performed with particles that are not infective at all. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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CE-microreactor-CE-MS/MS for protein analysis
Schoenherr, Regine M.; Ye, Mingliang; Vannatta, Michael
2008-01-01
We present a proof-of-principle for a fully automated bottom-up approach to protein characterization. Proteins are first separated by capillary electrophoresis. A pepsin microreactor is incorporated into the distal end of this capillary. Peptides formed in the reactor are transferred to a second capillary, where they are separated by capillary electrophoresis and characterized by mass spectrometry. While peptides generated from one digestion are being separated in the second capillary, the next protein fraction undergoes digestion in the microreactor. The migration time in the first dimension capillary is characteristic of the protein while migration time in the second dimension is characteristic of the peptide. Spot capacity for the two-dimensional separation is 590. A MS/MS analysis of a mixture of cytochrome C and myoglobin generated Mascot MOWSE scores of 107 for cytochrome C and 58 for myoglobin. The sequence coverages were 48% and 22%, respectively. PMID:17295444
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The carnegie protein trap library: a versatile tool for Drosophila developmental studies.
Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D; Nystul, Todd G; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E; Murphy, Terence D; Levis, Robert W; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A; Spradling, Allan C
2007-03-01
Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.
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The Bologna Annotation Resource (BAR 3.0): improving protein functional annotation.
Profiti, Giuseppe; Martelli, Pier Luigi; Casadio, Rita
2017-07-03
BAR 3.0 updates our server BAR (Bologna Annotation Resource) for predicting protein structural and functional features from sequence. We increase data volume, query capabilities and information conveyed to the user. The core of BAR 3.0 is a graph-based clustering procedure of UniProtKB sequences, following strict pairwise similarity criteria (sequence identity ≥40% with alignment coverage ≥90%). Each cluster contains the available annotation downloaded from UniProtKB, GO, PFAM and PDB. After statistical validation, GO terms and PFAM domains are cluster-specific and annotate new sequences entering the cluster after satisfying similarity constraints. BAR 3.0 includes 28 869 663 sequences in 1 361 773 clusters, of which 22.2% (22 241 661 sequences) and 47.4% (24 555 055 sequences) have at least one validated GO term and one PFAM domain, respectively. 1.4% of the clusters (36% of all sequences) include PDB structures and the cluster is associated to a hidden Markov model that allows building template-target alignment suitable for structural modeling. Some other 3 399 026 sequences are singletons. BAR 3.0 offers an improved search interface, allowing queries by UniProtKB-accession, Fasta sequence, GO-term, PFAM-domain, organism, PDB and ligand/s. When evaluated on the CAFA2 targets, BAR 3.0 largely outperforms our previous version and scores among state-of-the-art methods. BAR 3.0 is publicly available and accessible at http://bar.biocomp.unibo.it/bar3. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Modifications in trypsin digestion protocol for increasing the efficiency and coverage.
Syal, Kirtimaan; Tadala, Raghu
2015-01-01
Standard trypsin digestion protocol of proteins followed by MALDI-MS analysis has been realized as an important tool for the identification and characterization of proteins. In this article, we proposed the elimination of the step of 'staining/de-staining of gel pieces' in in-gel digestion protocol in order to improve the efficiency of trypsin digestion. Coomassie dye is known to interfere with digestion of proteins by trypsin and the procedure of staining-de-staining could result in loss of photoaffinity probe, post translational modifications and catalytic activities of enzymes. Further, we studied parameters like hydrophobicity and isoelectric point, and attempted to quantitatively relate it to the efficiency of trypsin digestion. We suggest that properties of proteins should be considered and trypsin digestion protocol should be appropriately modified as per sequence and other information.
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Salisbury, Joseph P; Sîrbulescu, Ruxandra F; Moran, Benjamin M; Auclair, Jared R; Zupanc, Günther K H; Agar, Jeffrey N
2015-03-11
The brown ghost knifefish (Apteronotus leptorhynchus) is a weakly electric teleost fish of particular interest as a versatile model system for a variety of research areas in neuroscience and biology. The comprehensive information available on the neurophysiology and neuroanatomy of this organism has enabled significant advances in such areas as the study of the neural basis of behavior, the development of adult-born neurons in the central nervous system and their involvement in the regeneration of nervous tissue, as well as brain aging and senescence. Despite substantial scientific interest in this species, no genomic resources are currently available. Here, we report the de novo assembly and annotation of the A. leptorhynchus transcriptome. After evaluating several trimming and transcript reconstruction strategies, de novo assembly using Trinity uncovered 42,459 unique contigs containing at least a partial protein-coding sequence based on alignment to a reference set of known Actinopterygii sequences. As many as 11,847 of these contigs contained full or near-full length protein sequences, providing broad coverage of the proteome. A variety of non-coding RNA sequences were also identified and annotated, including conserved long intergenic non-coding RNA and other long non-coding RNA observed previously to be expressed in adult zebrafish (Danio rerio) brain, as well as a variety of miRNA, snRNA, and snoRNA. Shotgun proteomics confirmed translation of open reading frames from over 2,000 transcripts, including alternative splice variants. Assignment of tandem mass spectra was greatly improved by use of the assembly compared to databases of sequences from closely related organisms. The assembly and raw reads have been deposited at DDBJ/EMBL/GenBank under the accession number GBKR00000000. Tandem mass spectrometry data is available via ProteomeXchange with identifier PXD001285. Presented here is the first release of an annotated de novo transcriptome assembly from Apteronotus leptorhynchus, providing a broad overview of RNA expressed in central nervous system tissue. The assembly, which includes substantial coverage of a wide variety of both protein coding and non-coding transcripts, will allow the development of better tools to understand the mechanisms underlying unique characteristics of the knifefish model system, such as their tremendous regenerative capacity and negligible brain senescence.
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Sequencing Larger Intact Proteins (30-70 kDa) with Activated Ion Electron Transfer Dissociation
NASA Astrophysics Data System (ADS)
Riley, Nicholas M.; Westphall, Michael S.; Coon, Joshua J.
2018-01-01
The analysis of intact proteins via mass spectrometry can offer several benefits to proteome characterization, although the majority of top-down experiments focus on proteoforms in a relatively low mass range (<30 kDa). Recent studies have focused on improving the analysis of larger intact proteins (up to 75 kDa), but they have also highlighted several challenges to be addressed. One major hurdle is the efficient dissociation of larger protein ions, which often to do not yield extensive fragmentation via conventional tandem MS methods. Here we describe the first application of activated ion electron transfer dissociation (AI-ETD) to proteins in the 30-70 kDa range. AI-ETD leverages infrared photo-activation concurrent to ETD reactions to improve sequence-informative product ion generation. This method generates more product ions and greater sequence coverage than conventional ETD, higher-energy collisional dissociation (HCD), and ETD combined with supplemental HCD activation (EThcD). Importantly, AI-ETD provides the most thorough protein characterization for every precursor ion charge state investigated in this study, making it suitable as a universal fragmentation method in top-down experiments. Additionally, we highlight several acquisition strategies that can benefit characterization of larger proteins with AI-ETD, including combination of spectra from multiple ETD reaction times for a given precursor ion, multiple spectral acquisitions of the same precursor ion, and combination of spectra from two different dissociation methods (e.g., AI-ETD and HCD). In all, AI-ETD shows great promise as a method for dissociating larger intact protein ions as top-down proteomics continues to advance into larger mass ranges. [Figure not available: see fulltext.
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snpAD: An ancient DNA genotype caller.
Prüfer, Kay
2018-06-21
The study of ancient genomes can elucidate the evolutionary past. However, analyses are complicated by base-modifications in ancient DNA molecules that result in errors in DNA sequences. These errors are particularly common near the ends of sequences and pose a challenge for genotype calling. I describe an iterative method that estimates genotype frequencies and errors along sequences to allow for accurate genotype calling from ancient sequences. The implementation of this method, called snpAD, performs well on high-coverage ancient data, as shown by simulations and by subsampling the data of a high-coverage Neandertal genome. Although estimates for low-coverage genomes are less accurate, I am able to derive approximate estimates of heterozygosity from several low-coverage Neandertals. These estimates show that low heterozygosity, compared to modern humans, was common among Neandertals. The C ++ code of snpAD is freely available at http://bioinf.eva.mpg.de/snpAD/. Supplementary data are available at Bioinformatics online.
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2011-01-01
Background One of the key goals of oak genomics research is to identify genes of adaptive significance. This information may help to improve the conservation of adaptive genetic variation and the management of forests to increase their health and productivity. Deep-coverage large-insert genomic libraries are a crucial tool for attaining this objective. We report herein the construction of a BAC library for Quercus robur, its characterization and an analysis of BAC end sequences. Results The EcoRI library generated consisted of 92,160 clones, 7% of which had no insert. Levels of chloroplast and mitochondrial contamination were below 3% and 1%, respectively. Mean clone insert size was estimated at 135 kb. The library represents 12 haploid genome equivalents and, the likelihood of finding a particular oak sequence of interest is greater than 99%. Genome coverage was confirmed by PCR screening of the library with 60 unique genetic loci sampled from the genetic linkage map. In total, about 20,000 high-quality BAC end sequences (BESs) were generated by sequencing 15,000 clones. Roughly 5.88% of the combined BAC end sequence length corresponded to known retroelements while ab initio repeat detection methods identified 41 additional repeats. Collectively, characterized and novel repeats account for roughly 8.94% of the genome. Further analysis of the BESs revealed 1,823 putative genes suggesting at least 29,340 genes in the oak genome. BESs were aligned with the genome sequences of Arabidopsis thaliana, Vitis vinifera and Populus trichocarpa. One putative collinear microsyntenic region encoding an alcohol acyl transferase protein was observed between oak and chromosome 2 of V. vinifera. Conclusions This BAC library provides a new resource for genomic studies, including SSR marker development, physical mapping, comparative genomics and genome sequencing. BES analysis provided insight into the structure of the oak genome. These sequences will be used in the assembly of a future genome sequence for oak. PMID:21645357
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Hill, Ryan C.; Wither, Matthew J.; Nemkov, Travis; Barrett, Alexander; D'Alessandro, Angelo; Dzieciatkowska, Monika; Hansen, Kirk C.
2015-01-01
Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827. PMID:25948757
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Naseer, Muhammad Imran; Rasool, Mahmood; Jan, Mohammed M; Chaudhary, Adeel G; Pushparaj, Peter Natesan; Abuzenadah, Adel M; Al-Qahtani, Mohammad H
2016-12-15
PGAP2 (Post-GPI Attachment to Proteins 2) gene is involved in lipid remodeling steps of Glycosylphosphatidylinositol (GPI)-anchor maturation. At the surface of the cell this gene is required for proper expression of GPI-anchored proteins. Hyperphosphatasia with mental retardation syndrome-3 is an autosomal recessive disorder usually characterized by severe mental retardation. Mutations in the PGAP2 gene cause hyperphosphatasia mental retardation syndrome-3. We have identified a large consanguineous family from Saudi origin segregating developmental delay, intellectual disability, epilepsy and microcephaly. Whole exome sequencing with 100× coverage was performed on two affected siblings of the family. Data analysis in the patient revealed a novel missense mutation c.191C>T in PGAP2 gene resulting in Alanine to Valine substitution (Ala64Val). The mutation was reconfirmed and validated by subsequent Sanger sequencing method. The mutation was ruled out in 100 unrelated healthy controls. We suggest that this pathogenic mutation disrupts the proper function of the gene proteins resulting in the disease state. Copyright © 2016 Elsevier B.V. All rights reserved.
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Indexcov: fast coverage quality control for whole-genome sequencing.
Pedersen, Brent S; Collins, Ryan L; Talkowski, Michael E; Quinlan, Aaron R
2017-11-01
The BAM and CRAM formats provide a supplementary linear index that facilitates rapid access to sequence alignments in arbitrary genomic regions. Comparing consecutive entries in a BAM or CRAM index allows one to infer the number of alignment records per genomic region for use as an effective proxy of sequence depth in each genomic region. Based on these properties, we have developed indexcov, an efficient estimator of whole-genome sequencing coverage to rapidly identify samples with aberrant coverage profiles, reveal large-scale chromosomal anomalies, recognize potential batch effects, and infer the sex of a sample. Indexcov is available at https://github.com/brentp/goleft under the MIT license. © The Authors 2017. Published by Oxford University Press.
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Assessing the Relationship of Ancient and Modern Populations
Schraiber, Joshua G.
2018-01-01
Genetic material sequenced from ancient samples is revolutionizing our understanding of the recent evolutionary past. However, ancient DNA is often degraded, resulting in low coverage, error-prone sequencing. Several solutions exist to this problem, ranging from simple approach, such as selecting a read at random for each site, to more complicated approaches involving genotype likelihoods. In this work, we present a novel method for assessing the relationship of an ancient sample with a modern population, while accounting for sequencing error and postmortem damage by analyzing raw reads from multiple ancient individuals simultaneously. We show that, when analyzing SNP data, it is better to sequence more ancient samples to low coverage: two samples sequenced to 0.5× coverage provide better resolution than a single sample sequenced to 2× coverage. We also examined the power to detect whether an ancient sample is directly ancestral to a modern population, finding that, with even a few high coverage individuals, even ancient samples that are very slightly diverged from the modern population can be detected with ease. When we applied our approach to European samples, we found that no ancient samples represent direct ancestors of modern Europeans. We also found that, as shown previously, the most ancient Europeans appear to have had the smallest effective population sizes, indicating a role for agriculture in modern population growth. PMID:29167200
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Zhang, Xi; Miller, Keith W.
2015-01-01
The challenge in high-quality membrane proteomics is all about sample preparation prior to HPLC, and the cell-to-protein step poses a long-standing bottleneck. Traditional protein extraction methods apply ionic or poly-disperse detergents, harsh denaturation, and repeated protein/peptide precipitation/resolubilization afterward, but suffer low yield, low reproducibility, and low sequence coverage. Contrary to attempts to subdue, we resolved this challenge by providing proteins nature-and-activity-promoting conditions throughout preparation. Using 285-kDa hetero-pentameric human GABA type A receptor overexpressed in HEK293 as a model, we describe a n-dodecyl-β-d-maltopyranoside/cholesteryl hemisuccinate (DDM/CHS)-based affinity purification method, that produced active receptors, supported protease activity, and allowed high performance with both in-gel and direct gel-free proteomic analyses—without detergent removal. Unlike conventional belief that detergents must be removed before HPLC MS, the high-purity low-dose nonionic detergent DDM did not interfere with peptides, and obviated removal or desalting. Sonication or dropwise addition of detergent robustly solubilized over 90% of membrane pellets. The purification conditions were comparable to those applied in successful crystallizations of most membrane proteins. These results enabled streamlined proteomics of human synaptic membrane proteins, and more importantly, allowed directly coupling proteomics with crystallography to characterize both static and dynamic structures of membrane proteins in crystallization pipelines. PMID:25473089
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Neville, B. Anne; Sheridan, Paul O.; Harris, Hugh M. B.; Coughlan, Simone; Flint, Harry J.; Duncan, Sylvia H.; Jeffery, Ian B.; Claesson, Marcus J.; Ross, R. Paul; Scott, Karen P.; O'Toole, Paul W.
2013-01-01
Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ28. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes. PMID:23935906
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Circulation of Endemic Type 2 Vaccine-Derived Poliovirus in Egypt from 1983 to 1993
Yang, Chen-Fu; Naguib, Tary; Yang, Su-Ju; Nasr, Eman; Jorba, Jaume; Ahmed, Nahed; Campagnoli, Ray; van der Avoort, Harrie; Shimizu, Hiroyuki; Yoneyama, Tetsuo; Miyamura, Tatsuo; Pallansch, Mark; Kew, Olen
2003-01-01
From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5′ untranslated region (5′ UTR) and noncapsid- 3′ UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide. PMID:12857906
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Circulation of endemic type 2 vaccine-derived poliovirus in Egypt from 1983 to 1993.
Yang, Chen-Fu; Naguib, Tary; Yang, Su-Ju; Nasr, Eman; Jorba, Jaume; Ahmed, Nahed; Campagnoli, Ray; van der Avoort, Harrie; Shimizu, Hiroyuki; Yoneyama, Tetsuo; Miyamura, Tatsuo; Pallansch, Mark; Kew, Olen
2003-08-01
From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5' untranslated region (5' UTR) and noncapsid- 3' UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide.
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Identification of a Herbal Powder by Deoxyribonucleic Acid Barcoding and Structural Analyses.
Sheth, Bhavisha P; Thaker, Vrinda S
2015-10-01
Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. To identify a herbal powder obtained from a herbalist in the local vicinity of Rajkot, Gujarat, using deoxyribonucleic acid (DNA) barcoding and molecular tools. The DNA was extracted from a herbal powder and selected Cassia species, followed by the polymerase chain reaction (PCR) and sequencing of the rbcL barcode locus. Thereafter the sequences were subjected to National Center for Biotechnology Information (NCBI) basic local alignment search tool (BLAST) analysis, followed by the protein three-dimension structure determination of the rbcL protein from the herbal powder and Cassia species namely Cassia fistula, Cassia tora and Cassia javanica (sequences obtained in the present study), Cassia Roxburghii, and Cassia abbreviata (sequences retrieved from Genbank). Further, the multiple and pairwise structural alignment were carried out in order to identify the herbal powder. The nucleotide sequences obtained from the selected species of Cassia were submitted to Genbank (Accession No. JX141397, JX141405, JX141420). The NCBI BLAST analysis of the rbcL protein from the herbal powder showed an equal sequence similarity (with reference to different parameters like E value, maximum identity, total score, query coverage) to C. javanica and C. roxburghii. In order to solve the ambiguities of the BLAST result, a protein structural approach was implemented. The protein homology models obtained in the present study were submitted to the protein model database (PM0079748-PM0079753). The pairwise structural alignment of the herbal powder (as template) and C. javanica and C. roxburghii (as targets individually) revealed a close similarity of the herbal powder with C. javanica. A strategy as used here, incorporating the integrated use of DNA barcoding and protein structural analyses could be adopted, as a novel rapid and economic procedure, especially in cases when protein coding loci are considered. Authentic identification of plants is essential for exploiting their medicinal properties as well as to stop the adulteration and malpractices with the trade of the same. A herbal powder was obtained from a herbalist in the local vicinity of Rajkot, Gujarat. An integrated approach using DNA barcoding and structural analyses was carried out to identify the herbal powder. The herbal powder was identified as Cassia javanica L.
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Launch mission summary and sequence of events Telesat-F(anik-D1)/Delta-164
NASA Technical Reports Server (NTRS)
1982-01-01
The launch vehicle, spacecraft, and mission are summarized. Launch window information, vehicle telemetry coverage, real time data flow, telemetry coverage by station, selected trajectory information, and a brief sequence of flight events are included.
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Backman, Samuel; Norlén, Olov; Eriksson, Barbro; Skogseid, Britt; Stålberg, Peter; Crona, Joakim
2017-02-01
Mutations affecting the mechanistic target of rapamycin (MTOR) signalling pathway are frequent in human cancer and have been identified in up to 15% of pancreatic neuroendocrine tumours (NETs). Grade A evidence supports the efficacy of MTOR inhibition with everolimus in pancreatic NETs. Although a significant proportion of patients experience disease stabilization, only a minority will show objective tumour responses. It has been proposed that genomic mutations resulting in activation of MTOR signalling could be used to predict sensitivity to everolimus. Patients with NETs that underwent treatment with everolimus at our Institution were identified and those with available tumour tissue were selected for further analysis. Targeted next-generation sequencing (NGS) was used to re-sequence 22 genes that were selected on the basis of documented involvement in the MTOR signalling pathway or in the tumourigenesis of gastroenterpancreatic NETs. Radiological responses were documented using Response Evaluation Criteria in Solid Tumours. Six patients were identified, one had a partial response and four had stable disease. Sequencing of tumour tissue resulted in a median sequence depth of 667.1 (range=404-1301) with 1-fold coverage of 95.9-96.5% and 10-fold coverage of 87.6-92.2%. A total of 494 genetic variants were discovered, four of which were identified as pathogenic. All pathogenic variants were validated using Sanger sequencing and were found exclusively in menin 1 (MEN1) and death domain associated protein (DAXX) genes. No mutations in the MTOR pathway-related genes were observed. Targeted NGS is a feasible method with high diagnostic yield for genetic characterization of pancreatic NETs. A potential association between mutations in NETs and response to everolimus should be investigated by future studies. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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NASA Astrophysics Data System (ADS)
Anderson, Lissa C.; Håkansson, Maria; Walse, Björn; Nilsson, Carol L.
2017-09-01
Structural technologies are an essential component in the design of precision therapeutics. Precision medicine entails the development of therapeutics directed toward a designated target protein, with the goal to deliver the right drug to the right patient at the right time. In the field of oncology, protein structural variants are often associated with oncogenic potential. In a previous proteogenomic screen of patient-derived glioblastoma (GBM) tumor materials, we identified a sequence variant of human mitochondrial branched-chain amino acid aminotransferase 2 as a putative factor of resistance of GBM to standard-of-care-treatments. The enzyme generates glutamate, which is neurotoxic. To elucidate structural coordinates that may confer altered substrate binding or activity of the variant BCAT2 T186R, a 45 kDa protein, we applied combined ETD and CID top-down mass spectrometry in a LC-FT-ICR MS at 21 T, and X-Ray crystallography in the study of both the variant and non-variant intact proteins. The combined ETD/CID fragmentation pattern allowed for not only extensive sequence coverage but also confident localization of the amino acid variant to its position in the sequence. The crystallographic experiments confirmed the hypothesis generated by in silico structural homology modeling, that the Lys59 side-chain of BCAT2 may repulse the Arg186 in the variant protein (PDB code: 5MPR), leading to destabilization of the protein dimer and altered enzyme kinetics. Taken together, the MS and novel 3D structural data give us reason to further pursue BCAT2 T186R as a precision drug target in GBM. [Figure not available: see fulltext.
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PDBe: Protein Data Bank in Europe
Velankar, S.; Alhroub, Y.; Best, C.; Caboche, S.; Conroy, M. J.; Dana, J. M.; Fernandez Montecelo, M. A.; van Ginkel, G.; Golovin, A.; Gore, S. P.; Gutmanas, A.; Haslam, P.; Hendrickx, P. M. S.; Heuson, E.; Hirshberg, M.; John, M.; Lagerstedt, I.; Mir, S.; Newman, L. E.; Oldfield, T. J.; Patwardhan, A.; Rinaldi, L.; Sahni, G.; Sanz-García, E.; Sen, S.; Slowley, R.; Suarez-Uruena, A.; Swaminathan, G. J.; Symmons, M. F.; Vranken, W. F.; Wainwright, M.; Kleywegt, G. J.
2012-01-01
The Protein Data Bank in Europe (PDBe; pdbe.org) is a partner in the Worldwide PDB organization (wwPDB; wwpdb.org) and as such actively involved in managing the single global archive of biomacromolecular structure data, the PDB. In addition, PDBe develops tools, services and resources to make structure-related data more accessible to the biomedical community. Here we describe recently developed, extended or improved services, including an animated structure-presentation widget (PDBportfolio), a widget to graphically display the coverage of any UniProt sequence in the PDB (UniPDB), chemistry- and taxonomy-based PDB-archive browsers (PDBeXplore), and a tool for interactive visualization of NMR structures, corresponding experimental data as well as validation and analysis results (Vivaldi). PMID:22110033
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Shaw, Jared B.; Robinson, Errol W.; Pasa-Tolic, Ljiljana
2016-02-16
We revisited the implementation of UVPD within the ICR cell of a FT-ICR mass spectrometer. UVPD performance characteristics were examined in the context of recent developments in the understanding of UVPD and in-cell tandem mass spectrometry. Efficient UVPD and photo-ECD of a model peptide and small protein within the ICR cell of a FT-ICR mass spectrometer are accomplished through appropriate modulation of laser pulse timing relative to ion magnetron motion and the potential applied to an ion optical element that photons impinge on. It is shown that UVPD yields efficient and extensive fragmentation resulting in excellent sequence coverage for modelmore » peptide and protein cations.« less
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Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W.; Howieson, John G.; Li, Chengdao
2013-01-01
Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species. PMID:23734219
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Yang, Huaan; Tao, Ye; Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W; Howieson, John G; Li, Chengdao
2013-01-01
Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species.
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Chen, Xiaochen; Li, Qiushi; Li, Ying; Qian, Jun; Han, Jianping
2015-01-01
The chloroplast genome (cp genome) of Aconitum barbatum var. puberulum was sequenced using the third-generation sequencing platform based on the single-molecule real-time (SMRT) sequencing approach. To our knowledge, this is the first reported complete cp genome of Aconitum, and we anticipate that it will have great value for phylogenetic studies of the Ranunculaceae family. In total, 23,498 CCS reads and 20,685,462 base pairs were generated, the mean read length was 880 bp, and the longest read was 2,261 bp. Genome coverage of 100% was achieved with a mean coverage of 132× and no gaps. The accuracy of the assembled genome is 99.973%; the assembly was validated using Sanger sequencing of six selected genes from the cp genome. The complete cp genome of A. barbatum var. puberulum is 156,749 bp in length, including a large single-copy region of 87,630 bp and a small single-copy region of 16,941 bp separated by two inverted repeats of 26,089 bp. The cp genome contains 130 genes, including 84 protein-coding genes, 34 tRNA genes and eight rRNA genes. Four forward, five inverted and eight tandem repeats were identified. According to the SSR analysis, the longest poly structure is a 20-T repeat. Our results presented in this paper will facilitate the phylogenetic studies and molecular authentication on Aconitum.
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Chen, Xiaochen; Li, Qiushi; Li, Ying; Qian, Jun; Han, Jianping
2015-01-01
The chloroplast genome (cp genome) of Aconitum barbatum var. puberulum was sequenced using the third-generation sequencing platform based on the single-molecule real-time (SMRT) sequencing approach. To our knowledge, this is the first reported complete cp genome of Aconitum, and we anticipate that it will have great value for phylogenetic studies of the Ranunculaceae family. In total, 23,498 CCS reads and 20,685,462 base pairs were generated, the mean read length was 880 bp, and the longest read was 2,261 bp. Genome coverage of 100% was achieved with a mean coverage of 132× and no gaps. The accuracy of the assembled genome is 99.973%; the assembly was validated using Sanger sequencing of six selected genes from the cp genome. The complete cp genome of A. barbatum var. puberulum is 156,749 bp in length, including a large single-copy region of 87,630 bp and a small single-copy region of 16,941 bp separated by two inverted repeats of 26,089 bp. The cp genome contains 130 genes, including 84 protein-coding genes, 34 tRNA genes and eight rRNA genes. Four forward, five inverted and eight tandem repeats were identified. According to the SSR analysis, the longest poly structure is a 20-T repeat. Our results presented in this paper will facilitate the phylogenetic studies and molecular authentication on Aconitum. PMID:25705213
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2011-01-01
Background HIV vaccine development must address the genetic diversity and plasticity of the virus that permits the presentation of diverse genetic forms to the immune system and subsequent escape from immune pressure. Assessment of potential HIV strain coverage by candidate T cell-based vaccines (whether natural sequence or computationally optimized products) is now a critical component in interpreting candidate vaccine suitability. Methods We have utilized an N-mer identity algorithm to represent T cell epitopes and explore potential coverage of the global HIV pandemic using natural sequences derived from candidate HIV vaccines. Breadth (the number of T cell epitopes generated) and depth (the variant coverage within a T cell epitope) analyses have been incorporated into the model to explore vaccine coverage requirements in terms of the number of discrete T cell epitopes generated. Results We show that when multiple epitope generation by a vaccine product is considered a far more nuanced appraisal of the potential HIV strain coverage of the vaccine product emerges. By considering epitope breadth and depth several important observations were made: (1) epitope breadth requirements to reach particular levels of vaccine coverage, even for natural sequence-based vaccine products is not necessarily an intractable problem for the immune system; (2) increasing the valency (number of T cell epitope variants present) of vaccine products dramatically decreases the epitope requirements to reach particular coverage levels for any epidemic; (3) considering multiple-hit models (more than one exact epitope match with an incoming HIV strain) places a significantly higher requirement upon epitope breadth in order to reach a given level of coverage, to the point where low valency natural sequence based products would not practically be able to generate sufficient epitopes. Conclusions When HIV vaccine sequences are compared against datasets of potential incoming viruses important metrics such as the minimum epitope count required to reach a desired level of coverage can be easily calculated. We propose that such analyses can be applied early in the planning stages and during the execution phase of a vaccine trial to explore theoretical and empirical suitability of a vaccine product to a particular epidemic setting. PMID:22152192
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Enhanced sequencing coverage with digital droplet multiple displacement amplification
Sidore, Angus M.; Lan, Freeman; Lim, Shaun W.; Abate, Adam R.
2016-01-01
Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing. PMID:26704978
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NASA Astrophysics Data System (ADS)
Sharrar, A.; Diamond, S.; Butterfield, C.; Starr, E.; Thomas, B. C.; Banfield, J. F.
2016-12-01
Soils are extremely heterogeneous and diverse microbial habitats. The distribution of microbes and their metabolic functions in soil is important for carbon and nitrogen cycling and overall ecosystem functioning. Little is known about biogeographical patterns of microbes in soil and how they relate to different environmental gradients. To address this topic, we have obtained metagenomes from 86 soil samples taken at the Eel River Critical Zone Observatory and a nearby meadow in the northern California Angelo Coast Range Reserve over a period of two years. These samples span a variety of environmental parameters, including depth, relief, time since last rainfall, and proximity to root carbon inputs. Phylogenetic diversity and community overlap between these samples was analyzed using ribosomal protein sequence identity and scaffold coverage. Independent of genomic bins, de-replicated ribosomal protein sequences were used to identify thousands of unique organisms between datasets. Despite environmental differences, a subset of these organisms were found in every sample. The dominant phyla in this cosmopolitan subset were Proteobacteria, Actinobacteria, Gemmatimonadetes, Verrucomicrobia, Acidobacteria, and Rokubacteria. Archaea and Nitrospirae were particularly prevalent at depths below 30cm. Patterns of ribosomal protein scaffold coverage across samples was used to compare the similarity of the microbial communities. We found that location was the greatest predictor of community similarity, followed by depth. In a subset of 60 samples taken in the same meadow, sampling plot was a greater predictor of community similarity than time since last rainfall in the same year. In addition, genome-independent analyses revealed differences in the metabolic potential for functions involved in nitrogen and carbon metabolism across environmental gradients. This study will further our understanding of soil biogeography and its relationship to overall biogeochemical cycling.
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Estimating genotype error rates from high-coverage next-generation sequence data.
Wall, Jeffrey D; Tang, Ling Fung; Zerbe, Brandon; Kvale, Mark N; Kwok, Pui-Yan; Schaefer, Catherine; Risch, Neil
2014-11-01
Exome and whole-genome sequencing studies are becoming increasingly common, but little is known about the accuracy of the genotype calls made by the commonly used platforms. Here we use replicate high-coverage sequencing of blood and saliva DNA samples from four European-American individuals to estimate lower bounds on the error rates of Complete Genomics and Illumina HiSeq whole-genome and whole-exome sequencing. Error rates for nonreference genotype calls range from 0.1% to 0.6%, depending on the platform and the depth of coverage. Additionally, we found (1) no difference in the error profiles or rates between blood and saliva samples; (2) Complete Genomics sequences had substantially higher error rates than Illumina sequences had; (3) error rates were higher (up to 6%) for rare or unique variants; (4) error rates generally declined with genotype quality (GQ) score, but in a nonlinear fashion for the Illumina data, likely due to loss of specificity of GQ scores greater than 60; and (5) error rates increased with increasing depth of coverage for the Illumina data. These findings, especially (3)-(5), suggest that caution should be taken in interpreting the results of next-generation sequencing-based association studies, and even more so in clinical application of this technology in the absence of validation by other more robust sequencing or genotyping methods. © 2014 Wall et al.; Published by Cold Spring Harbor Laboratory Press.
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Pitteri, Sharon J.; Chrisman, Paul A.; McLuckey, Scott A.
2005-01-01
In this study, the electron-transfer dissociation (ETD) behavior of cations derived from 27 different peptides (22 of which are tryptic peptides) has been studied in a 3D quadrupole ion trap mass spectrometer. Ion/ion reactions between peptide cations and nitrobenzene anions have been examined at both room temperature and in an elevated temperature bath gas environment to form ETD product ions. From the peptides studied, the ETD sequence coverage tends to be inversely related to peptide size. At room temperature, very high sequence coverage (~100%) was observed for small peptides (≤7 amino acids). For medium-sized peptides composed of 8–11 amino acids, the average sequence coverage was 46%. Larger peptides with 14 or more amino acids yielded an average sequence coverage of 23%. Elevated-temperature ETD provided increased sequence coverage over room-temperature experiments for the peptides of greater than 7 residues, giving an average of 67% for medium-sized peptides and 63% for larger peptides. Percent ETD, a measure of the extent of electron transfer, has also been calculated for the peptides and also shows an inverse relation with peptide size. Bath gas temperature does not have a consistent effect on percent ETD, however. For the tryptic peptides, fragmentation is localized at the ends of the peptides suggesting that the distribution of charge within the peptide may play an important role in determining fragmentation sites. A triply protonated peptide has also been studied and shows behavior similar to the doubly charged peptides. These preliminary results suggest that for a given charge state there is a maximum size for which high sequence coverage is obtained and that increasing the bath gas temperature can increase this maximum. PMID:16131079
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Calvano, Cosima Damiana; van der Werf, Inez Dorothé; Sabbatini, Luigia; Palmisano, Francesco
2015-05-01
The simultaneous identification of lipids and proteins by matrix assisted laser desorption ionization-mass spectrometry (MALDI-MS) after direct on-plate processing of micro-samples supported on colloidal graphite is demonstrated. Taking advantages of large surface area and thermal conductivity, graphite provided an ideal substrate for on-plate proteolysis and lipid extraction. Indeed proteins could be efficiently digested on-plate within 15 min, providing sequence coverages comparable to those obtained by conventional in-solution overnight digestion. Interestingly, detection of hydrophilic phosphorylated peptides could be easily achieved without any further enrichment step. Furthermore, lipids could be simultaneously extracted/identified without any additional treatment/processing step as demonstrated for model complex samples such as milk and egg. The present approach is simple, efficient, of large applicability and offers great promise for protein and lipid identification in very small samples. Copyright © 2015 Elsevier B.V. All rights reserved.
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Isolation and initial structural characterization of a 27 kDa protein from Zingiber officinale
NASA Astrophysics Data System (ADS)
Rasheed, Saima; Malik, Shoaib Ahmad; Falke, Sven; Arslan, Ali; Fazel, Ramin; Schlüter, Hartmut; Betzel, Christian; Choudhary, M. Iqbal
2018-03-01
Zingiber officinale Roscoe (Ginger) is a widely used traditional medicinal plant (for different ailments such as arthritis, constipation, and hypertension). This article describes the isolation and characterization of a so far unknown protein from ginger rhizomes applying ion exchange, affinity, size-exclusion chromatography, small angle X-ray scattering (SAXS), and mass spectrometry techniques. One-dimensional Coomassie-stained SDS-PAGE was performed under non-reducing conditions, showing one band corresponding to approx. 27 kDa. Dynamic light scattering (DLS) analysis of the protein solution revealed monodispersity and a monomeric state of the purified protein. Circular dichroism (CD) spectroscopy strongly indicated a β-sheet-rich protein, and disordered regions. MALDI-TOF-MS, and LC-MS/MS analysis resulted in the identification of 27.29 kDa protein, having 32.13% and 25.34% sequence coverage with Zingipain-1 and 2, respectively. The monomeric state and molecular weight were verified by small angle X-ray scattering (SAXS) studies. An elongated ab-initio model was calculated based on the scattering intensity distribution.
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Electrochemistry-Assisted Top-Down Characterization of Disulfide-Containing Proteins
Zhang, Yun; Cui, Weidong; Zhang, Hao; Dewald, Howard D.; Chen, Hao
2013-01-01
Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then online ionized into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs. 73 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs. 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research. PMID:22448817
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Electrochemistry-assisted top-down characterization of disulfide-containing proteins.
Zhang, Yun; Cui, Weidong; Zhang, Hao; Dewald, Howard D; Chen, Hao
2012-04-17
Covalent disulfide bond linkage in a protein represents an important challenge for mass spectrometry (MS)-based top-down protein structure analysis as it reduces the backbone cleavage efficiency for MS/MS dissociation. This study presents a strategy for solving this critical issue via integrating electrochemistry (EC) online with a top-down MS approach. In this approach, proteins undergo electrolytic reduction in an electrochemical cell to break disulfide bonds and then undergo online ionization into gaseous ions for analysis by electron-capture dissociation (ECD) and collision-induced dissociation (CID). The electrochemical reduction of proteins allows one to remove disulfide bond constraints and also leads to increased charge numbers of the resulting protein ions. As a result, sequence coverage was significantly enhanced, as exemplified by β-lactoglobulin A (24 vs 75 backbone cleavages before and after electrolytic reduction, respectively) and lysozyme (5 vs 66 backbone cleavages before and after electrolytic reduction, respectively). This methodology is fast and does not need chemical reductants, which would have an important impact in high-throughput proteomics research.
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Landscape of genomic diversity and trait discovery in soybean.
Valliyodan, Babu; Dan Qiu; Patil, Gunvant; Zeng, Peng; Huang, Jiaying; Dai, Lu; Chen, Chengxuan; Li, Yanjun; Joshi, Trupti; Song, Li; Vuong, Tri D; Musket, Theresa A; Xu, Dong; Shannon, J Grover; Shifeng, Cheng; Liu, Xin; Nguyen, Henry T
2016-03-31
Cultivated soybean [Glycine max (L.) Merr.] is a primary source of vegetable oil and protein. We report a landscape analysis of genome-wide genetic variation and an association study of major domestication and agronomic traits in soybean. A total of 106 soybean genomes representing wild, landraces, and elite lines were re-sequenced at an average of 17x depth with a 97.5% coverage. Over 10 million high-quality SNPs were discovered, and 35.34% of these have not been previously reported. Additionally, 159 putative domestication sweeps were identified, which includes 54.34 Mbp (4.9%) and 4,414 genes; 146 regions were involved in artificial selection during domestication. A genome-wide association study of major traits including oil and protein content, salinity, and domestication traits resulted in the discovery of novel alleles. Genomic information from this study provides a valuable resource for understanding soybean genome structure and evolution, and can also facilitate trait dissection leading to sequencing-based molecular breeding.
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Landscape of genomic diversity and trait discovery in soybean
Valliyodan, Babu; Dan Qiu; Patil, Gunvant; Zeng, Peng; Huang, Jiaying; Dai, Lu; Chen, Chengxuan; Li, Yanjun; Joshi, Trupti; Song, Li; Vuong, Tri D.; Musket, Theresa A.; Xu, Dong; Shannon, J. Grover; Shifeng, Cheng; Liu, Xin; Nguyen, Henry T.
2016-01-01
Cultivated soybean [Glycine max (L.) Merr.] is a primary source of vegetable oil and protein. We report a landscape analysis of genome-wide genetic variation and an association study of major domestication and agronomic traits in soybean. A total of 106 soybean genomes representing wild, landraces, and elite lines were re-sequenced at an average of 17x depth with a 97.5% coverage. Over 10 million high-quality SNPs were discovered, and 35.34% of these have not been previously reported. Additionally, 159 putative domestication sweeps were identified, which includes 54.34 Mbp (4.9%) and 4,414 genes; 146 regions were involved in artificial selection during domestication. A genome-wide association study of major traits including oil and protein content, salinity, and domestication traits resulted in the discovery of novel alleles. Genomic information from this study provides a valuable resource for understanding soybean genome structure and evolution, and can also facilitate trait dissection leading to sequencing-based molecular breeding. PMID:27029319
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Nonpareil 3: Fast Estimation of Metagenomic Coverage and Sequence Diversity.
Rodriguez-R, Luis M; Gunturu, Santosh; Tiedje, James M; Cole, James R; Konstantinidis, Konstantinos T
2018-01-01
Estimations of microbial community diversity based on metagenomic data sets are affected, often to an unknown degree, by biases derived from insufficient coverage and reference database-dependent estimations of diversity. For instance, the completeness of reference databases cannot be generally estimated since it depends on the extant diversity sampled to date, which, with the exception of a few habitats such as the human gut, remains severely undersampled. Further, estimation of the degree of coverage of a microbial community by a metagenomic data set is prohibitively time-consuming for large data sets, and coverage values may not be directly comparable between data sets obtained with different sequencing technologies. Here, we extend Nonpareil, a database-independent tool for the estimation of coverage in metagenomic data sets, to a high-performance computing implementation that scales up to hundreds of cores and includes, in addition, a k -mer-based estimation as sensitive as the original alignment-based version but about three hundred times as fast. Further, we propose a metric of sequence diversity ( N d ) derived directly from Nonpareil curves that correlates well with alpha diversity assessed by traditional metrics. We use this metric in different experiments demonstrating the correlation with the Shannon index estimated on 16S rRNA gene profiles and show that N d additionally reveals seasonal patterns in marine samples that are not captured by the Shannon index and more precise rankings of the magnitude of diversity of microbial communities in different habitats. Therefore, the new version of Nonpareil, called Nonpareil 3, advances the toolbox for metagenomic analyses of microbiomes. IMPORTANCE Estimation of the coverage provided by a metagenomic data set, i.e., what fraction of the microbial community was sampled by DNA sequencing, represents an essential first step of every culture-independent genomic study that aims to robustly assess the sequence diversity present in a sample. However, estimation of coverage remains elusive because of several technical limitations associated with high computational requirements and limiting statistical approaches to quantify diversity. Here we described Nonpareil 3, a new bioinformatics algorithm that circumvents several of these limitations and thus can facilitate culture-independent studies in clinical or environmental settings, independent of the sequencing platform employed. In addition, we present a new metric of sequence diversity based on rarefied coverage and demonstrate its use in communities from diverse ecosystems.
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Zhang, Ai-bing; Feng, Jie; Ward, Robert D; Wan, Ping; Gao, Qiang; Wu, Jun; Zhao, Wei-zhong
2012-01-01
Species identification via DNA barcodes is contributing greatly to current bioinventory efforts. The initial, and widely accepted, proposal was to use the protein-coding cytochrome c oxidase subunit I (COI) region as the standard barcode for animals, but recently non-coding internal transcribed spacer (ITS) genes have been proposed as candidate barcodes for both animals and plants. However, achieving a robust alignment for non-coding regions can be problematic. Here we propose two new methods (DV-RBF and FJ-RBF) to address this issue for species assignment by both coding and non-coding sequences that take advantage of the power of machine learning and bioinformatics. We demonstrate the value of the new methods with four empirical datasets, two representing typical protein-coding COI barcode datasets (neotropical bats and marine fish) and two representing non-coding ITS barcodes (rust fungi and brown algae). Using two random sub-sampling approaches, we demonstrate that the new methods significantly outperformed existing Neighbor-joining (NJ) and Maximum likelihood (ML) methods for both coding and non-coding barcodes when there was complete species coverage in the reference dataset. The new methods also out-performed NJ and ML methods for non-coding sequences in circumstances of potentially incomplete species coverage, although then the NJ and ML methods performed slightly better than the new methods for protein-coding barcodes. A 100% success rate of species identification was achieved with the two new methods for 4,122 bat queries and 5,134 fish queries using COI barcodes, with 95% confidence intervals (CI) of 99.75-100%. The new methods also obtained a 96.29% success rate (95%CI: 91.62-98.40%) for 484 rust fungi queries and a 98.50% success rate (95%CI: 96.60-99.37%) for 1094 brown algae queries, both using ITS barcodes.
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Dong, Jiajia; Vicente, Natallia; Chintauan-Marquier, Ioana C; Ramadi, Cahyo; Dettai, Agnès; Robillard, Tony
2017-05-15
In the present study, we report the high-coverage complete mitochondrial genome (mitogenome) of the cricket Cardiodactylus muiri Otte, 2007. The mitogenome was sequenced using a long-PCR approach on an Ion Torrent Personal Genome Machine (PGM) for next generation sequencing technology. The total length of the amplified mitogenome is 16,328 bp, representing 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one noncoding region (D-loop region). The new sets of long-PCR primers reported here are invaluable resources for future comparative evolutionary genomic studies in Orthopteran insects. The new mitogenome sequence is compared with published cricket mitogenomes. In the taxonomic part, we present new records for the species and describe life-history traits, habitat and male calling song of the species; based on observation of new material, the species Cardiodactylus buru Gorochov & Robillard, 2014 is synonymized under C. muiri.
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Haplotype estimation using sequencing reads.
Delaneau, Olivier; Howie, Bryan; Cox, Anthony J; Zagury, Jean-François; Marchini, Jonathan
2013-10-03
High-throughput sequencing technologies produce short sequence reads that can contain phase information if they span two or more heterozygote genotypes. This information is not routinely used by current methods that infer haplotypes from genotype data. We have extended the SHAPEIT2 method to use phase-informative sequencing reads to improve phasing accuracy. Our model incorporates the read information in a probabilistic model through base quality scores within each read. The method is primarily designed for high-coverage sequence data or data sets that already have genotypes called. One important application is phasing of single samples sequenced at high coverage for use in medical sequencing and studies of rare diseases. Our method can also use existing panels of reference haplotypes. We tested the method by using a mother-father-child trio sequenced at high-coverage by Illumina together with the low-coverage sequence data from the 1000 Genomes Project (1000GP). We found that use of phase-informative reads increases the mean distance between switch errors by 22% from 274.4 kb to 328.6 kb. We also used male chromosome X haplotypes from the 1000GP samples to simulate sequencing reads with varying insert size, read length, and base error rate. When using short 100 bp paired-end reads, we found that using mixtures of insert sizes produced the best results. When using longer reads with high error rates (5-20 kb read with 4%-15% error per base), phasing performance was substantially improved. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
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Jarecki, Jessica L.; Frey, Brian L.; Smith, Lloyd M.; Stretton, Antony O.
2011-01-01
Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to discover peptides in extracts of the large parasitic nematode Ascaris suum. This required the assembly of a new database of known and predicted peptides. In addition to those already sequenced, peptides were either previously predicted to be processed from precursor proteins identified in an A. suum library of expressed sequence tags (ESTs), or newly predicted from a library of A. suum genome survey sequences (GSSs). The predicted MS/MS fragmentation patterns of this collection of real and putative peptides were compared with the actual fragmentation patterns found in the MS/MS spectra of peptides fractionated by MS; this enabled individual peptides to be sequenced. Many previously identified peptides were found, and 21 novel peptides were discovered. Thus, this approach is very useful, despite the fact that the available GSS database is still preliminary, having only 1X coverage. PMID:21524146
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CORNAS: coverage-dependent RNA-Seq analysis of gene expression data without biological replicates.
Low, Joel Z B; Khang, Tsung Fei; Tammi, Martti T
2017-12-28
In current statistical methods for calling differentially expressed genes in RNA-Seq experiments, the assumption is that an adjusted observed gene count represents an unknown true gene count. This adjustment usually consists of a normalization step to account for heterogeneous sample library sizes, and then the resulting normalized gene counts are used as input for parametric or non-parametric differential gene expression tests. A distribution of true gene counts, each with a different probability, can result in the same observed gene count. Importantly, sequencing coverage information is currently not explicitly incorporated into any of the statistical models used for RNA-Seq analysis. We developed a fast Bayesian method which uses the sequencing coverage information determined from the concentration of an RNA sample to estimate the posterior distribution of a true gene count. Our method has better or comparable performance compared to NOISeq and GFOLD, according to the results from simulations and experiments with real unreplicated data. We incorporated a previously unused sequencing coverage parameter into a procedure for differential gene expression analysis with RNA-Seq data. Our results suggest that our method can be used to overcome analytical bottlenecks in experiments with limited number of replicates and low sequencing coverage. The method is implemented in CORNAS (Coverage-dependent RNA-Seq), and is available at https://github.com/joel-lzb/CORNAS .
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Lara Carrillo, John Alejandro; Fierro Medina, Ricardo; Manríquez Rocha, Juan; Bustos Bustos, Erika; Insuasty Cepeda, Diego Sebastián; García Castañeda, Javier Eduardo; Rivera Monroy, Zuly Jenny
2017-11-14
In order to obtain gold electrode surfaces modified with Human Papillomavirus L1 protein (HPV L1)-derived peptides, two sequences, SPINNTKPHEAR and YIK, were chosen. Both have been recognized by means of sera from patients infected with HPV. The molecules, Fc-Ahx-SPINNTKPHEAR, Ac-C- Ahx -(Fc)KSPINNTKPHEAR, Ac-C- Ahx -SPINNTKPHEAR(Fc)K, C- Ahx -SPINNTKPHEAR, and (YIK)₂- Ahx -C, were designed, synthesized, and characterized. Our results suggest that peptides derived from the SPINNTKPHEAR sequence, containing ferrocene and cysteine residues, are not stable and not adequate for electrode surface modification. The surface of polycrystalline gold electrodes was modified with the peptides C-Ahx-SPINNTKPHEAR or (YIK)₂-Ahx-C through self-assembly. The modified polycrystalline gold electrodes were characterized via infrared spectroscopy and electrochemical measurements. The thermodynamic parameters, surface coverage factor, and medium pH effect were determined for these surfaces. The results indicate that surface modification depends on the peptide sequence (length, amino acid composition, polyvalence, etc.). The influence of antipeptide antibodies on the voltammetric response of the modified electrode was evaluated by comparing results obtained with pre-immune and post-immune serum samples.
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Thatcher, Louise F; Williams, Angela H; Garg, Gagan; Buck, Sally-Anne G; Singh, Karam B
2016-11-03
Pathogenic members of the Fusarium oxysporum species complex are responsible for vascular wilt disease on many important crops including legumes, where they can be one of the most destructive disease causing necrotrophic fungi. We previously developed a model legume-infecting pathosystem based on the reference legume Medicago truncatula and a pathogenic F. oxysporum forma specialis (f. sp.) medicaginis (Fom). To dissect the molecular pathogenicity arsenal used by this root-infecting pathogen, we sequenced its transcriptome during infection of a susceptible and resistant host accession. High coverage RNA-Seq of Fom infected root samples harvested from susceptible (DZA315) or resistant (A17) M. truncatula seedlings at early or later stages of infection (2 or 7 days post infection (dpi)) and from vegetative (in vitro) samples facilitated the identification of unique and overlapping sets of in planta differentially expressed genes. This included enrichment, particularly in DZA315 in planta up-regulated datasets, for proteins associated with sugar, protein and plant cell wall metabolism, membrane transport, nutrient uptake and oxidative processes. Genes encoding effector-like proteins were identified, including homologues of the F. oxysporum f. sp. lycopersici Secreted In Xylem (SIX) proteins, and several novel candidate effectors based on predicted secretion, small protein size and high in-planta induced expression. The majority of the effector candidates contain no known protein domains but do share high similarity to predicted proteins predominantly from other F. oxysporum ff. spp. as well as other Fusaria (F. solani, F. fujikori, F. verticilloides, F. graminearum and F. pseudograminearum), and from another wilt pathogen of the same class, a Verticillium species. Overall, this suggests these novel effector candidates may play important roles in Fusaria and wilt pathogen virulence. Combining high coverage in planta RNA-Seq with knowledge of fungal pathogenicity protein features facilitated the identification of differentially expressed pathogenicity associated genes and novel effector candidates expressed during infection of a resistant or susceptible M. truncatula host. The knowledge from this first in depth in planta transcriptome sequencing of any F. oxysporum ff. spp. pathogenic on legumes will facilitate the dissection of Fusarium wilt pathogenicity mechanisms on many important legume crops.
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Mass spectrometry compatible surfactant for optimized in-gel protein digestion.
Saveliev, Sergei V; Woodroofe, Carolyn C; Sabat, Grzegorz; Adams, Christopher M; Klaubert, Dieter; Wood, Keith; Urh, Marjeta
2013-01-15
Identification of proteins resolved by SDS-PAGE depends on robust in-gel protein digestion and efficient peptide extraction, requirements that are often difficult to achieve. A lengthy and laborious procedure is an additional challenge of protein identification in gel. We show here that with the use of the mass spectrometry compatible surfactant sodium 3-((1-(furan-2-yl)undecyloxy)carbonylamino)propane-1-sulfonate, the challenges of in-gel protein digestion are effectively addressed. Peptide quantitation based on stable isotope labeling showed that the surfactant induced 1.5-2 fold increase in peptide recovery. Consequently, protein sequence coverage was increased by 20-30%, on average, and the number of identified proteins saw a substantial boost. The surfactant also accelerated the digestion process. Maximal in-gel digestion was achieved in as little as one hour, depending on incubation temperature, and peptides were readily recovered from gel eliminating the need for postdigestion extraction. This study shows that the surfactant provides an efficient means of improving protein identification in gel and streamlining the in-gel digestion procedure requiring no extra handling steps or special equipment.
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High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic.
Sealfon, Rachel; Gire, Stephen; Ellis, Crystal; Calderwood, Stephen; Qadri, Firdausi; Hensley, Lisa; Kellis, Manolis; Ryan, Edward T; LaRocque, Regina C; Harris, Jason B; Sabeti, Pardis C
2012-09-11
Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.
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G23D: Online tool for mapping and visualization of genomic variants on 3D protein structures.
Solomon, Oz; Kunik, Vered; Simon, Amos; Kol, Nitzan; Barel, Ortal; Lev, Atar; Amariglio, Ninette; Somech, Raz; Rechavi, Gidi; Eyal, Eran
2016-08-26
Evaluation of the possible implications of genomic variants is an increasingly important task in the current high throughput sequencing era. Structural information however is still not routinely exploited during this evaluation process. The main reasons can be attributed to the partial structural coverage of the human proteome and the lack of tools which conveniently convert genomic positions, which are the frequent output of genomic pipelines, to proteins and structure coordinates. We present G23D, a tool for conversion of human genomic coordinates to protein coordinates and protein structures. G23D allows mapping of genomic positions/variants on evolutionary related (and not only identical) protein three dimensional (3D) structures as well as on theoretical models. By doing so it significantly extends the space of variants for which structural insight is feasible. To facilitate interpretation of the variant consequence, pathogenic variants, functional sites and polymorphism sites are displayed on protein sequence and structure diagrams alongside the input variants. G23D also provides modeling of the mutant structure, analysis of intra-protein contacts and instant access to functional predictions and predictions of thermo-stability changes. G23D is available at http://www.sheba-cancer.org.il/G23D . G23D extends the fraction of variants for which structural analysis is applicable and provides better and faster accessibility for structural data to biologists and geneticists who routinely work with genomic information.
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2011-01-01
Background Integration of genomic variation with phenotypic information is an effective approach for uncovering genotype-phenotype associations. This requires an accurate identification of the different types of variation in individual genomes. Results We report the integration of the whole genome sequence of a single Holstein Friesian bull with data from single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) array technologies to determine a comprehensive spectrum of genomic variation. The performance of resequencing SNP detection was assessed by combining SNPs that were identified to be either in identity by descent (IBD) or in copy number variation (CNV) with results from SNP array genotyping. Coding insertions and deletions (indels) were found to be enriched for size in multiples of 3 and were located near the N- and C-termini of proteins. For larger indels, a combination of split-read and read-pair approaches proved to be complementary in finding different signatures. CNVs were identified on the basis of the depth of sequenced reads, and by using SNP and CGH arrays. Conclusions Our results provide high resolution mapping of diverse classes of genomic variation in an individual bovine genome and demonstrate that structural variation surpasses sequence variation as the main component of genomic variability. Better accuracy of SNP detection was achieved with little loss of sensitivity when algorithms that implemented mapping quality were used. IBD regions were found to be instrumental for calculating resequencing SNP accuracy, while SNP detection within CNVs tended to be less reliable. CNV discovery was affected dramatically by platform resolution and coverage biases. The combined data for this study showed that at a moderate level of sequencing coverage, an ensemble of platforms and tools can be applied together to maximize the accurate detection of sequence and structural variants. PMID:22082336
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The Carnegie Protein Trap Library: A Versatile Tool for Drosophila Developmental Studies
Buszczak, Michael; Paterno, Shelley; Lighthouse, Daniel; Bachman, Julia; Planck, Jamie; Owen, Stephenie; Skora, Andrew D.; Nystul, Todd G.; Ohlstein, Benjamin; Allen, Anna; Wilhelm, James E.; Murphy, Terence D.; Levis, Robert W.; Matunis, Erika; Srivali, Nahathai; Hoskins, Roger A.; Spradling, Allan C.
2007-01-01
Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600–900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions. PMID:17194782
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De Novo Peptide Sequencing: Deep Mining of High-Resolution Mass Spectrometry Data.
Islam, Mohammad Tawhidul; Mohamedali, Abidali; Fernandes, Criselda Santan; Baker, Mark S; Ranganathan, Shoba
2017-01-01
High resolution mass spectrometry has revolutionized proteomics over the past decade, resulting in tremendous amounts of data in the form of mass spectra, being generated in a relatively short span of time. The mining of this spectral data for analysis and interpretation though has lagged behind such that potentially valuable data is being overlooked because it does not fit into the mold of traditional database searching methodologies. Although the analysis of spectra by de novo sequences removes such biases and has been available for a long period of time, its uptake has been slow or almost nonexistent within the scientific community. In this chapter, we propose a methodology to integrate de novo peptide sequencing using three commonly available software solutions in tandem, complemented by homology searching, and manual validation of spectra. This simplified method would allow greater use of de novo sequencing approaches and potentially greatly increase proteome coverage leading to the unearthing of valuable insights into protein biology, especially of organisms whose genomes have been recently sequenced or are poorly annotated.
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FY11 Report on Metagenome Analysis using Pathogen Marker Libraries
DOE Office of Scientific and Technical Information (OSTI.GOV)
Gardner, Shea N.; Allen, Jonathan E.; McLoughlin, Kevin S.
2011-06-02
A method, sequence library, and software suite was invented to rapidly assess whether any member of a pre-specified list of threat organisms or their near neighbors is present in a metagenome. The system was designed to handle mega- to giga-bases of FASTA-formatted raw sequence reads from short or long read next generation sequencing platforms. The approach is to pre-calculate a viral and a bacterial "Pathogen Marker Library" (PML) containing sub-sequences specific to pathogens or their near neighbors. A list of expected matches comparing every bacterial or viral genome against the PML sequences is also pre-calculated. To analyze a metagenome, readsmore » are compared to the PML, and observed PML-metagenome matches are compared to the expected PML-genome matches, and the ratio of observed relative to expected matches is reported. In other words, a 3-way comparison among the PML, metagenome, and existing genome sequences is used to quickly assess which (if any) species included in the PML is likely to be present in the metagenome, based on available sequence data. Our tests showed that the species with the most PML matches correctly indicated the organism sequenced for empirical metagenomes consisting of a cultured, relatively pure isolate. These runs completed in 1 minute to 3 hours on 12 CPU (1 thread/CPU), depending on the metagenome and PML. Using more threads on the same number of CPU resulted in speed improvements roughly proportional to the number of threads. Simulations indicated that detection sensitivity depends on both sequencing coverage levels for a species and the size of the PML: species were correctly detected even at ~0.003x coverage by the large PMLs, and at ~0.03x coverage by the smaller PMLs. Matches to true positive species were 3-4 orders of magnitude higher than to false positives. Simulations with short reads (36 nt and ~260 nt) showed that species were usually detected for metagenome coverage above 0.005x and coverage in the PML above 0.05x, and detection probability appears to be a function of both coverages. Multiple species could be detected simultaneously in a simulated low-coverage, complex metagenome, and the largest PML gave no false negative species and no false positive genera. The presence of multiple species was predicted in a complex metagenome from a human gut microbiome with 1.9 GB of short reads (75 nt); the species predicted were reasonable gut flora and no biothreat agents were detected, showing the feasibility of PML analysis of empirical complex metagenomes.« less
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Shaw, Jared B; Robinson, Errol W; Paša-Tolić, Ljiljana
2016-03-15
We revisited the implementation of 193 nm ultraviolet photodissociation (UVPD) within the ion cyclotron resonance (ICR) cell of a Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer. UVPD performance characteristics were examined in the context of recent developments in the understanding of UVPD and in-cell tandem mass spectrometry. Efficient UVPD and photo-ECD of a model peptide and proteins within the ICR cell of a FT-ICR mass spectrometer are accomplished through appropriate modulation of laser pulse timing, relative to ion magnetron motion and the potential applied to an ion optical element upon which photons impinge. It is shown that UVPD yields efficient and extensive fragmentation, resulting in excellent sequence coverage for model peptide and protein cations.
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Combining functional and structural genomics to sample the essential Burkholderia structome.
Baugh, Loren; Gallagher, Larry A; Patrapuvich, Rapatbhorn; Clifton, Matthew C; Gardberg, Anna S; Edwards, Thomas E; Armour, Brianna; Begley, Darren W; Dieterich, Shellie H; Dranow, David M; Abendroth, Jan; Fairman, James W; Fox, David; Staker, Bart L; Phan, Isabelle; Gillespie, Angela; Choi, Ryan; Nakazawa-Hewitt, Steve; Nguyen, Mary Trang; Napuli, Alberto; Barrett, Lynn; Buchko, Garry W; Stacy, Robin; Myler, Peter J; Stewart, Lance J; Manoil, Colin; Van Voorhis, Wesley C
2013-01-01
The genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite. We experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an "ortholog rescue" strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail. This collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request.
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Boguś, M I; Wieloch, W; Ligęza-Żuber, M
2017-02-01
Coronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC-MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva-1. Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.
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NASA Astrophysics Data System (ADS)
Ningrum, R. A.; Santoso, A.; Herawati, N.
2017-05-01
Human interferon alpha2a (hIFNα2a) is a therapeutic protein that used in cancer and hepatitis B/C therapy. The main problem of using hIFNα-2a is its short elimination half life due to its low molecular weight. Development of higher molecular weight protein by albumin fusion technology is a rational strategy to solve the problem. In our previous research we constructed an open reading frame (ORF) encoding hIFNα2a-human serum albumin (HSA) fusion protein that expressed in Pichia pastoris (P. pastoris) protease deficient strain SMD1168. This research was performed to overproduce, purify and characterize the fusion protein. To overproduce the protein, cultivation was performed in buffered complex medium containing glyserol (BMGY) for 24 h and protein overproduction was applied in buffered complex medium containing methanol (BMMY) for 48 hours at 30°C. The fusion protein was purified by blue sepharose affinity chromatography. Molecular weight characterization by SDS PAGE corresponds with its theoretical size, 85 kDa. Western blot analysis demonstrated that the fusion protein was recognized by anti hIFNα2 and anti HSA monoclonal antibody as well. Amino acid sequence of the fusion protein was determined by LC MS/MS2 mass spectrometry with trypsin as proteolitic enzyme. There were three fragments that identified as hIFNα2a and seven fragments that identified as HSA. Total identified amino acids were 150 residues with 20% coverage from total residues. To conclude, hIFNα2a-HSA fusion protein was overproduced, purified and characterized. Characterization based on molecular weight, antibody recognition and amino acid sequence confirmed that the fusion protein has correct identity as theoretically thought.
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O'Brien, Heath E; Gong, Yunchen; Fung, Pauline; Wang, Pauline W; Guttman, David S
2011-01-01
Next-generation genomic technology has both greatly accelerated the pace of genome research as well as increased our reliance on draft genome sequences. While groups such as the Genomics Standards Consortium have made strong efforts to promote genome standards there is a still a general lack of uniformity among published draft genomes, leading to challenges for downstream comparative analyses. This lack of uniformity is a particular problem when using standard draft genomes that frequently have large numbers of low-quality sequencing tracts. Here we present a proposal for an "enhanced-quality draft" genome that identifies at least 95% of the coding sequences, thereby effectively providing a full accounting of the genic component of the genome. Enhanced-quality draft genomes are easily attainable through a combination of small- and large-insert next-generation, paired-end sequencing. We illustrate the generation of an enhanced-quality draft genome by re-sequencing the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola 1448A (Pph 1448A), which has a published, closed genome sequence of 5.93 Mbp. We use a combination of Illumina paired-end and mate-pair sequencing, and surprisingly find that de novo assemblies with 100x paired-end coverage and mate-pair sequencing with as low as low as 2-5x coverage are substantially better than assemblies based on higher coverage. The rapid and low-cost generation of large numbers of enhanced-quality draft genome sequences will be of particular value for microbial diagnostics and biosecurity, which rely on precise discrimination of potentially dangerous clones from closely related benign strains.
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Shannon C.K. Straub; Mark Fishbein; Tatyana Livshult; Zachary Foster; Matthew Parks; Kevin Weitemier; Richard C. Cronn; Aaron Liston
2011-01-01
Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in...
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Breaking Lander-Waterman’s Coverage Bound
Nashta-ali, Damoun; Motahari, Seyed Abolfazl; Hosseinkhalaj, Babak
2016-01-01
Lander-Waterman’s coverage bound establishes the total number of reads required to cover the whole genome of size G bases. In fact, their bound is a direct consequence of the well-known solution to the coupon collector’s problem which proves that for such genome, the total number of bases to be sequenced should be O(G ln G). Although the result leads to a tight bound, it is based on a tacit assumption that the set of reads are first collected through a sequencing process and then are processed through a computation process, i.e., there are two different machines: one for sequencing and one for processing. In this paper, we present a significant improvement compared to Lander-Waterman’s result and prove that by combining the sequencing and computing processes, one can re-sequence the whole genome with as low as O(G) sequenced bases in total. Our approach also dramatically reduces the required computational power for the combined process. Simulation results are performed on real genomes with different sequencing error rates. The results support our theory predicting the log G improvement on coverage bound and corresponding reduction in the total number of bases required to be sequenced. PMID:27806058
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Sepúlveda, Nuno; Campino, Susana G; Assefa, Samuel A; Sutherland, Colin J; Pain, Arnab; Clark, Taane G
2013-02-26
The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data.
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Fu, Yong-Bi; Peterson, Gregory W; Dong, Yibo
2016-04-07
Genotyping-by-sequencing (GBS) has emerged as a useful genomic approach for exploring genome-wide genetic variation. However, GBS commonly samples a genome unevenly and can generate a substantial amount of missing data. These technical features would limit the power of various GBS-based genetic and genomic analyses. Here we present software called IgCoverage for in silico evaluation of genomic coverage through GBS with an individual or pair of restriction enzymes on one sequenced genome, and report a new set of 21 restriction enzyme combinations that can be applied to enhance GBS applications. These enzyme combinations were developed through an application of IgCoverage on 22 plant, animal, and fungus species with sequenced genomes, and some of them were empirically evaluated with different runs of Illumina MiSeq sequencing in 12 plant species. The in silico analysis of 22 organisms revealed up to eight times more genome coverage for the new combinations consisted of pairing four- or five-cutter restriction enzymes than the commonly used enzyme combination PstI + MspI. The empirical evaluation of the new enzyme combination (HinfI + HpyCH4IV) in 12 plant species showed 1.7-6 times more genome coverage than PstI + MspI, and 2.3 times more genome coverage in dicots than monocots. Also, the SNP genotyping in 12 Arabidopsis and 12 rice plants revealed that HinfI + HpyCH4IV generated 7 and 1.3 times more SNPs (with 0-16.7% missing observations) than PstI + MspI, respectively. These findings demonstrate that these novel enzyme combinations can be utilized to increase genome sampling and improve SNP genotyping in various GBS applications. Copyright © 2016 Fu et al.
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Nóbrega de Sousa, Taís; Carvalho, Luzia Helena; Alves de Brito, Cristiana Ferreira
2011-01-01
The dependence of Plasmodium vivax on invasion mediated by Duffy binding protein (DBP) makes this protein a prime candidate for development of a vaccine. However, the development of a DBP-based vaccine might be hampered by the high variability of the protein ligand (DBP(II)), known to bias the immune response toward a specific DBP variant. Here, the hypothesis being investigated is that the analysis of the worldwide DBP(II) sequences will allow us to determine the minimum number of haplotypes (MNH) to be included in a DBP-based vaccine of broad coverage. For that, all DBP(II) sequences available were compiled and MNH was based on the most frequent nonsynonymous single nucleotide polymorphisms, the majority mapped on B and T cell epitopes. A preliminary analysis of DBP(II) genetic diversity from eight malaria-endemic countries estimated that a number between two to six DBP haplotypes (17 in total) would target at least 50% of parasite population circulating in each endemic region. Aiming to avoid region-specific haplotypes, we next analyzed the MNH that broadly cover worldwide parasite population. The results demonstrated that seven haplotypes would be required to cover around 60% of DBP(II) sequences available. Trying to validate these selected haplotypes per country, we found that five out of the eight countries will be covered by the MNH (67% of parasite populations, range 48-84%). In addition, to identify related subgroups of DBP(II) sequences we used a Bayesian clustering algorithm. The algorithm grouped all DBP(II) sequences in six populations that were independent of geographic origin, with ancestral populations present in different proportions in each country. In conclusion, in this first attempt to undertake a global analysis about DBP(II) variability, the results suggest that the development of DBP-based vaccine should consider multi-haplotype strategies; otherwise a putative P. vivax vaccine may not target some parasite populations.
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Prosdocimi, Francisco; Souto, Helena Magarinos; Ruschi, Piero Angeli; Furtado, Carolina; Jennings, W Bryan
2016-09-01
The genome of the versicoloured emerald hummingbird (Amazilia versicolor) was partially sequenced in one-sixth of an Illumina HiSeq lane. The mitochondrial genome was assembled using MIRA and MITObim software, yielding a circular molecule of 16,861 bp in length and deposited in GenBank under the accession number KF624601. The mitogenome contained 13 protein-coding genes, 22 transfer tRNAs, 2 ribosomal RNAs and 1 non-coding control region. The molecule was assembled using 21,927 sequencing reads of 100 bp each, resulting in ∼130 × coverage of uniformly distributed reads along the genome. This is the forth mitochondrial genome described for this highly diverse family of birds and may benefit further phylogenetic, phylogeographic, population genetic and species delimitation studies of hummingbirds.
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Mutua, Martin Kavao; Kimani-Murage, Elizabeth; Ngomi, Nicholas; Ravn, Henrik; Mwaniki, Peter; Echoka, Elizabeth
2016-01-01
More efforts have been put in place to increase full immunization coverage rates in the last decade. Little is known about the levels and consequences of delaying or vaccinating children in different schedules. Vaccine effectiveness depends on the timing of its administration, and it is not optimal if given early, delayed or not given as recommended. Evidence of non-specific effects of vaccines is well documented and could be linked to timing and sequencing of immunization. This paper documents the levels of coverage, timing and sequencing of routine childhood vaccines. The study was conducted between 2007 and 2014 in two informal urban settlements in Nairobi. A total of 3856 children, aged 12-23 months and having a vaccination card seen were included in analysis. Vaccination dates recorded from the cards seen were used to define full immunization coverage, timeliness and sequencing. Proportions, medians and Kaplan-Meier curves were used to assess and describe the levels of full immunization coverage, vaccination delays and sequencing. The findings indicate that 67 % of the children were fully immunized by 12 months of age. Missing measles and third doses of polio and pentavalent vaccine were the main reason for not being fully immunized. Delays were highest for third doses of polio and pentavalent and measles. About 22 % of fully immunized children had vaccines in an out-of-sequence manner with 18 % not receiving pentavalent together with polio vaccine as recommended. Results show higher levels of missed opportunities and low coverage of routine childhood vaccinations given at later ages. New strategies are needed to enable health care providers and parents/guardians to work together to increase the levels of completion of all required vaccinations. In particular, more focus is needed on vaccines given in multiple doses (polio, pentavalent and pneumococcal conjugate vaccines).
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Doyle, Stephen R; Griffith, Ian S; Murphy, Nick P; Strugnell, Jan M
2015-01-01
The complete mitochondrial genome of the Eastern Rock lobster, Sagmariasus verreauxi, is reported for the first time. Using low-coverage, long read MiSeq next generation sequencing, we constructed and determined the mtDNA genome organization of the 15,470 bp sequence from two isolates from Eastern Tasmania, Australia and Northern New Zealand, and identified 46 polymorphic nucleotides between the two sequences. This genome sequence and its genetic polymorphisms will likely be useful in understanding the distribution and population connectivity of the Eastern Rock Lobster, and in the fisheries management of this commercially important species.
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Comparing de novo genome assembly: the long and short of it.
Narzisi, Giuseppe; Mishra, Bud
2011-04-29
Recent advances in DNA sequencing technology and their focal role in Genome Wide Association Studies (GWAS) have rekindled a growing interest in the whole-genome sequence assembly (WGSA) problem, thereby, inundating the field with a plethora of new formalizations, algorithms, heuristics and implementations. And yet, scant attention has been paid to comparative assessments of these assemblers' quality and accuracy. No commonly accepted and standardized method for comparison exists yet. Even worse, widely used metrics to compare the assembled sequences emphasize only size, poorly capturing the contig quality and accuracy. This paper addresses these concerns: it highlights common anomalies in assembly accuracy through a rigorous study of several assemblers, compared under both standard metrics (N50, coverage, contig sizes, etc.) as well as a more comprehensive metric (Feature-Response Curves, FRC) that is introduced here; FRC transparently captures the trade-offs between contigs' quality against their sizes. For this purpose, most of the publicly available major sequence assemblers--both for low-coverage long (Sanger) and high-coverage short (Illumina) reads technologies--are compared. These assemblers are applied to microbial (Escherichia coli, Brucella, Wolbachia, Staphylococcus, Helicobacter) and partial human genome sequences (Chr. Y), using sequence reads of various read-lengths, coverages, accuracies, and with and without mate-pairs. It is hoped that, based on these evaluations, computational biologists will identify innovative sequence assembly paradigms, bioinformaticists will determine promising approaches for developing "next-generation" assemblers, and biotechnologists will formulate more meaningful design desiderata for sequencing technology platforms. A new software tool for computing the FRC metric has been developed and is available through the AMOS open-source consortium.
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Lam, Maggie P Y; Lau, Edward; Siu, S O; Ng, Dominic C M; Kong, Ricky P W; Chiu, Philip C N; Yeung, William S B; Lo, Clive; Chu, Ivan K
2011-11-01
In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Minneci, Federico; Piovesan, Damiano; Cozzetto, Domenico; Jones, David T.
2013-01-01
To understand fully cell behaviour, biologists are making progress towards cataloguing the functional elements in the human genome and characterising their roles across a variety of tissues and conditions. Yet, functional information – either experimentally validated or computationally inferred by similarity – remains completely missing for approximately 30% of human proteins. FFPred was initially developed to bridge this gap by targeting sequences with distant or no homologues of known function and by exploiting clear patterns of intrinsic disorder associated with particular molecular activities and biological processes. Here, we present an updated and improved version, which builds on larger datasets of protein sequences and annotations, and uses updated component feature predictors as well as revised training procedures. FFPred 2.0 includes support vector regression models for the prediction of 442 Gene Ontology (GO) terms, which largely expand the coverage of the ontology and of the biological process category in particular. The GO term list mainly revolves around macromolecular interactions and their role in regulatory, signalling, developmental and metabolic processes. Benchmarking experiments on newly annotated proteins show that FFPred 2.0 provides more accurate functional assignments than its predecessor and the ProtFun server do; also, its assignments can complement information obtained using BLAST-based transfer of annotations, improving especially prediction in the biological process category. Furthermore, FFPred 2.0 can be used to annotate proteins belonging to several eukaryotic organisms with a limited decrease in prediction quality. We illustrate all these points through the use of both precision-recall plots and of the COGIC scores, which we recently proposed as an alternative numerical evaluation measure of function prediction accuracy. PMID:23717476
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Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing
2018-02-20
Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.
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Carss, Keren J; Arno, Gavin; Erwood, Marie; Stephens, Jonathan; Sanchis-Juan, Alba; Hull, Sarah; Megy, Karyn; Grozeva, Detelina; Dewhurst, Eleanor; Malka, Samantha; Plagnol, Vincent; Penkett, Christopher; Stirrups, Kathleen; Rizzo, Roberta; Wright, Genevieve; Josifova, Dragana; Bitner-Glindzicz, Maria; Scott, Richard H; Clement, Emma; Allen, Louise; Armstrong, Ruth; Brady, Angela F; Carmichael, Jenny; Chitre, Manali; Henderson, Robert H H; Hurst, Jane; MacLaren, Robert E; Murphy, Elaine; Paterson, Joan; Rosser, Elisabeth; Thompson, Dorothy A; Wakeling, Emma; Ouwehand, Willem H; Michaelides, Michel; Moore, Anthony T; Webster, Andrew R; Raymond, F Lucy
2017-01-05
Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in CHM in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease. Copyright © 2017. Published by Elsevier Inc.
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COACH: profile-profile alignment of protein families using hidden Markov models.
Edgar, Robert C; Sjölander, Kimmen
2004-05-22
Alignments of two multiple-sequence alignments, or statistical models of such alignments (profiles), have important applications in computational biology. The increased amount of information in a profile versus a single sequence can lead to more accurate alignments and more sensitive homolog detection in database searches. Several profile-profile alignment methods have been proposed and have been shown to improve sensitivity and alignment quality compared with sequence-sequence methods (such as BLAST) and profile-sequence methods (e.g. PSI-BLAST). Here we present a new approach to profile-profile alignment we call Comparison of Alignments by Constructing Hidden Markov Models (HMMs) (COACH). COACH aligns two multiple sequence alignments by constructing a profile HMM from one alignment and aligning the other to that HMM. We compare the alignment accuracy of COACH with two recently published methods: Yona and Levitt's prof_sim and Sadreyev and Grishin's COMPASS. On two sets of reference alignments selected from the FSSP database, we find that COACH is able, on average, to produce alignments giving the best coverage or the fewest errors, depending on the chosen parameter settings. COACH is freely available from www.drive5.com/lobster
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The salivary gland transcriptome of the eastern tree hole mosquito, Ochlerotatus triseriatus.
Calvo, Eric; Sanchez-Vargas, Irma; Kotsyfakis, Michalis; Favreau, Amanda J; Barbian, Kent D; Pham, Van M; Olson, Kenneth E; Ribeiro, José M C
2010-05-01
Saliva of blood-sucking arthropods contains a complex mixture of peptides that affect their host's hemostasis, inflammation, and immunity. These activities can also modify the site of pathogen delivery and increase disease transmission. Saliva also induces hosts to mount an antisaliva immune response that can lead to skin allergies or even anaphylaxis. Accordingly, knowledge of the salivary repertoire, or sialome, of a mosquito is useful to provide a knowledge platform to mine for novel pharmacological activities, to develop novel vaccine targets for vector-borne diseases, and to develop epidemiological markers of vector exposure and candidate desensitization vaccines. The mosquito Ochlerotatus triseriatus is a vector of La Crosse virus and produces allergy in humans. In this work, a total of 1,575 clones randomly selected from an adult female O. triseriatus salivary gland cDNA library was sequenced and used to assemble a database that yielded 731 clusters of related sequences, 560 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 159 protein sequences, 66 of which code for putative secreted proteins. Supplemental spreadsheets containing these data are available at http://exon.niaid.nih.gov/transcriptome/Ochlerotatus_triseriatus/S1/Ot-S1.xls and http://exon.niaid. nih.gov/transcriptome/Ochlerotatus_triseriatus/S2/Ot-S2.xls.
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Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum.
VanBuren, Robert; Bryant, Doug; Edger, Patrick P; Tang, Haibao; Burgess, Diane; Challabathula, Dinakar; Spittle, Kristi; Hall, Richard; Gu, Jenny; Lyons, Eric; Freeling, Michael; Bartels, Dorothea; Ten Hallers, Boudewijn; Hastie, Alex; Michael, Todd P; Mockler, Todd C
2015-11-26
Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a 'near-complete' draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.
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Hornshøj, Linda; Benn, Christine Stabell; Fernandes, Manuel; Rodrigues, Amabelia; Aaby, Peter; Fisker, Ane Bærent
2012-01-01
Objective The WHO aims for 90% coverage of the Expanded Program on Immunization (EPI), which in Guinea-Bissau included BCG vaccine at birth, three doses of diphtheria−tetanus−pertussis vaccine (DTP) and oral polio vaccine (OPV) at 6, 10 and 14 weeks and measles vaccine (MV) at 9 months when this study was conducted. The WHO assesses coverage by 12 months of age. The sequence of vaccines may have an effect on child mortality, but is not considered in official statistics or assessments of programme performance. We assessed vaccination coverage and frequency of out-of-sequence vaccinations by 12 and 24 months of age. Design Observational cohort study. Setting and participants The Bandim Health Project's (BHP) rural Health and Demographic Surveillance site covers 258 randomly selected villages in all regions of Guinea-Bissau. Villages are visited biannually and vaccination cards inspected to ascertain vaccination status. Between 2003 and 2009 vaccination status by 12 months of age was assessed for 5806 children aged 12–23 months; vaccination status by 24 months of age was assessed for 3792 children aged 24–35 months. Outcome measures Coverage of EPI vaccinations and frequency of out-of-sequence vaccinations. Results Half of 12-month-old children and 65% of 24-month-old children had completed all EPI vaccinations. Many children received vaccines out of sequence: by 12 months of age 54% of BCG-vaccinated children had received DTP with or before BCG and 28% of measles-vaccinated children had received DTP with or after MV. By 24 months of age the proportion of out-of-sequence vaccinations was 58% and 35%, respectively, for BCG and MV. Conclusions In rural Guinea-Bissau vaccination coverage by 12 months of age was low, but continued to increase beyond 12 months of age. More than half of all children received vaccinations out of sequence. This highlights the need to improve vaccination services. PMID:23166127
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Lessons for livestock genomics from genome and transcriptome sequencing in cattle and other mammals.
Taylor, Jeremy F; Whitacre, Lynsey K; Hoff, Jesse L; Tizioto, Polyana C; Kim, JaeWoo; Decker, Jared E; Schnabel, Robert D
2016-08-17
Decreasing sequencing costs and development of new protocols for characterizing global methylation, gene expression patterns and regulatory regions have stimulated the generation of large livestock datasets. Here, we discuss experiences in the analysis of whole-genome and transcriptome sequence data. We analyzed whole-genome sequence (WGS) data from 132 individuals from five canid species (Canis familiaris, C. latrans, C. dingo, C. aureus and C. lupus) and 61 breeds, three bison (Bison bison), 64 water buffalo (Bubalus bubalis) and 297 bovines from 17 breeds. By individual, data vary in extent of reference genome depth of coverage from 4.9X to 64.0X. We have also analyzed RNA-seq data for 580 samples representing 159 Bos taurus and Rattus norvegicus animals and 98 tissues. By aligning reads to a reference assembly and calling variants, we assessed effects of average depth of coverage on the actual coverage and on the number of called variants. We examined the identity of unmapped reads by assembling them and querying produced contigs against the non-redundant nucleic acids database. By imputing high-density single nucleotide polymorphism data on 4010 US registered Angus animals to WGS using Run4 of the 1000 Bull Genomes Project and assessing the accuracy of imputation, we identified misassembled reference sequence regions. We estimate that a 24X depth of coverage is required to achieve 99.5 % coverage of the reference assembly and identify 95 % of the variants within an individual's genome. Genomes sequenced to low average coverage (e.g., <10X) may fail to cover 10 % of the reference genome and identify <75 % of variants. About 10 % of genomic DNA or transcriptome sequence reads fail to align to the reference assembly. These reads include loci missing from the reference assembly and misassembled genes and interesting symbionts, commensal and pathogenic organisms. Assembly errors and a lack of annotation of functional elements significantly limit the utility of the current draft livestock reference assemblies. The Functional Annotation of Animal Genomes initiative seeks to annotate functional elements, while a 70X Pac-Bio assembly for cow is underway and may result in a significantly improved reference assembly.
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Chang, Vivian Y.; Federman, Noah; Martinez-Agosto, Julian; Tatishchev, Sergei F.; Nelson, Stanley F.
2014-01-01
Background Gastric adenocarcinoma is a rare diagnosis in childhood. A 14-year old male patient presented with metastatic gastric adenocarcinoma, and a strong family history of colon cancer. Clinical sequencing of CDH1 and APC were negative. Whole exome sequencing was therefore applied to capture the majority of protein-coding regions for the identification of single-nucleotide variants, small insertion/deletions, and copy number abnormalities in the patient’s germline as well as primary tumor. Materials and Methods DNA was extracted from the patient’s blood, primary tumor, and the unaffected mother’s blood. DNA libraries were constructed and sequenced on Illumina HiSeq2000. Data were post-processed using Picard and Samtools, then analyzed with the Genome Analysis Toolkit. Variants were annotated using an in-house Ensembl-based program. Copy number was assessed using ExomeCNV. Results Each sample was sequenced to a mean depth of coverage of greater than 120×. A rare non-synonymous coding SNV in TP53 was identified in the germline. There were 10 somatic cancer protein-damaging variants that were not observed in the unaffected mother genome. ExomeCNV comparing tumor to the patient’s germline, identified abnormal copy number, spanning 6,946 genes. Conclusion We present an unusual case of Li-Fraumeni detected by whole exome sequencing. There were also likely driver somatic mutations in the gastric adenocarcinoma. These results highlight the need for more thorough and broad scale germline and cancer analyses to accurately inform patients of inherited risk to cancer and to identify somatic mutations. PMID:23015295
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USDA-ARS?s Scientific Manuscript database
Copy number variations (CNVs) are large insertions, deletions or duplications in the genome that vary between members of a species and are known to affect a wide variety of phenotypic traits. In this study, we identified CNVs in a population of bulls using low coverage next-generation sequence data....
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Perez-Martinez, Angy P; Ong, Edison; Zhang, Lixin; Marrs, Carl F; He, Yongqun; Yang, Zhenhua
2017-11-01
H56/AERAS-456+IC31 (H56), composed of two early secretion proteins, Ag85B and ESAT-6, and a latency associated protein, Rv2660, and the IC31 Intercell adjuvant, is a new fusion subunit vaccine candidate designed to induce immunity against both new infection and reactivation of latent tuberculosis infection. Efficacy of subunit vaccines may be affected by the diversity of vaccine antigens among clinical strains and the extent of recognition by the diverse HLA molecules in the recipient population. Although a previous study showed the conservative nature of Ag85B- and ESAT-6-encoding genes, genetic diversity of Rv2660c that encodes RV2660 is largely unknown. The population coverage of H56 as a whole yet remains to be assessed. The present study was conducted to address these important knowledge gaps. DNA sequence analysis of Rv2660c found no variation among 83 of the 84 investigated clinical strains belonging to four genetic lineages. H56 was predicted to have as high as 99.6% population coverage in the South Africa population using the Immune Epitope Database (IEDB) Population Coverage Tool. Further comparison of H56 population coverage between South African Blacks and Caucasians based on the phenotypic frequencies of binding MHC Class I and Class II supertype alleles found that all of the nine MHC-I and six of eight MHC-II human leukocyte antigen (HLA) supertype alleles analyzed were significantly differentially expressed between the two subpopulations. This finding suggests the presence of race-specific functional binding motifs of MHC-I and MHC-II HLA alleles, which, in turn, highlights the importance of including diverse populations in vaccine clinical evaluation. In conclusion, H56 vaccine is predicted to have a promising population coverage in South Africa; this study demonstrates the utility of integrating comparative genomics and bioinformatics in bridging animal and clinical studies of novel TB vaccines. Copyright © 2017 Elsevier B.V. All rights reserved.
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Garamszegi, Sara; Franzosa, Eric A; Xia, Yu
2013-01-01
A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1) domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2) domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral-host interactions that are otherwise hidden in the traditional binary network, highlighting the power and necessity of high-resolution approaches in host-pathogen systems biology.
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Initial sequence and comparative analysis of the cat genome
Pontius, Joan U.; Mullikin, James C.; Smith, Douglas R.; Lindblad-Toh, Kerstin; Gnerre, Sante; Clamp, Michele; Chang, Jean; Stephens, Robert; Neelam, Beena; Volfovsky, Natalia; Schäffer, Alejandro A.; Agarwala, Richa; Narfström, Kristina; Murphy, William J.; Giger, Urs; Roca, Alfred L.; Antunes, Agostinho; Menotti-Raymond, Marilyn; Yuhki, Naoya; Pecon-Slattery, Jill; Johnson, Warren E.; Bourque, Guillaume; Tesler, Glenn; O’Brien, Stephen J.
2007-01-01
The genome sequence (1.9-fold coverage) of an inbred Abyssinian domestic cat was assembled, mapped, and annotated with a comparative approach that involved cross-reference to annotated genome assemblies of six mammals (human, chimpanzee, mouse, rat, dog, and cow). The results resolved chromosomal positions for 663,480 contigs, 20,285 putative feline gene orthologs, and 133,499 conserved sequence blocks (CSBs). Additional annotated features include repetitive elements, endogenous retroviral sequences, nuclear mitochondrial (numt) sequences, micro-RNAs, and evolutionary breakpoints that suggest historic balancing of translocation and inversion incidences in distinct mammalian lineages. Large numbers of single nucleotide polymorphisms (SNPs), deletion insertion polymorphisms (DIPs), and short tandem repeats (STRs), suitable for linkage or association studies were characterized in the context of long stretches of chromosome homozygosity. In spite of the light coverage capturing ∼65% of euchromatin sequence from the cat genome, these comparative insights shed new light on the tempo and mode of gene/genome evolution in mammals, promise several research applications for the cat, and also illustrate that a comparative approach using more deeply covered mammals provides an informative, preliminary annotation of a light (1.9-fold) coverage mammal genome sequence. PMID:17975172
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Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae.
Juozapaitis, Mindaugas; Serva, Andrius; Zvirbliene, Aurelija; Slibinskas, Rimantas; Staniulis, Juozas; Sasnauskas, Kestutis; Shiell, Brian J; Wang, Lin-Fa; Michalski, Wojtek P
2007-03-01
Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.
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Antigenic Drift Defines a New D4 Subgenotype of Measles Virus.
Muñoz-Alía, Miguel Ángel; Muller, Claude P; Russell, Stephen J
2017-06-01
The measles virus hemagglutinin (MeV-H) protein is the main target of protective neutralizing antibodies. Using a panel of monoclonal antibodies (MAbs) that recognize known major antigenic sites in MeV-H, we identified a D4 genotype variant that escapes neutralization by MAbs targeting the neutralizing epitope (NE) antigenic site. By site-directed mutagenesis, L249P was identified as the critical mutation disrupting the NE in this genotype D4 variant. Forty-two available D4 genotype gene sequences were subsequently analyzed and divided into 2 groups according to the presence or absence of the L249P MeV-H mutation. Further analysis of the MeV-N gene sequences of these 2 groups confirmed that they represent clearly definable, sequence-divergent D4 subgenotypes, which we named subgenotypes D4.1 and D4.2. The subgenotype D4.1 MeVs were isolated predominantly in Kenya and Ethiopia, whereas the MAb-resistant subgenotype D4.2 MeVs were isolated predominantly in France and Great Britain, countries with higher vaccine coverage rates. Interestingly, D4.2 subgenotype viruses showed a trend toward diminished susceptibility to neutralization by human sera pooled from approximately 60 to 80 North American donors. Escape from MAb neutralization may be a powerful epidemiological surveillance tool to monitor the evolution of new MeV subgenotypes. IMPORTANCE Measles virus is a paradigmatic RNA virus, as the antigenic composition of the vaccination has not needed to be updated since its discovery. The vaccine confers protection by inducing neutralizing antibodies that interfere with the function of the hemagglutinin protein. Viral strains are indistinguishable serologically, although characteristic nucleotide sequences differentiate 24 genotypes. In this work, we describe a distant evolutionary branch within genotype D4. Designated subgenotype D4.2, this virus is distinguishable by neutralization with vaccine-induced monoclonal antibodies that target the neutralizing epitope (NE). The subgenotype D4.2 viruses have a higher predominance in countries with intermediary levels of vaccine coverage. Our studies demonstrate that subgenotype D4.2 lacks epitopes associated with half of the known antigenic sites, which significantly impacts our understanding of measles virus evolution. Copyright © 2017 American Society for Microbiology.
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Antigenic Drift Defines a New D4 Subgenotype of Measles Virus
Muller, Claude P.
2017-01-01
ABSTRACT The measles virus hemagglutinin (MeV-H) protein is the main target of protective neutralizing antibodies. Using a panel of monoclonal antibodies (MAbs) that recognize known major antigenic sites in MeV-H, we identified a D4 genotype variant that escapes neutralization by MAbs targeting the neutralizing epitope (NE) antigenic site. By site-directed mutagenesis, L249P was identified as the critical mutation disrupting the NE in this genotype D4 variant. Forty-two available D4 genotype gene sequences were subsequently analyzed and divided into 2 groups according to the presence or absence of the L249P MeV-H mutation. Further analysis of the MeV-N gene sequences of these 2 groups confirmed that they represent clearly definable, sequence-divergent D4 subgenotypes, which we named subgenotypes D4.1 and D4.2. The subgenotype D4.1 MeVs were isolated predominantly in Kenya and Ethiopia, whereas the MAb-resistant subgenotype D4.2 MeVs were isolated predominantly in France and Great Britain, countries with higher vaccine coverage rates. Interestingly, D4.2 subgenotype viruses showed a trend toward diminished susceptibility to neutralization by human sera pooled from approximately 60 to 80 North American donors. Escape from MAb neutralization may be a powerful epidemiological surveillance tool to monitor the evolution of new MeV subgenotypes. IMPORTANCE Measles virus is a paradigmatic RNA virus, as the antigenic composition of the vaccination has not needed to be updated since its discovery. The vaccine confers protection by inducing neutralizing antibodies that interfere with the function of the hemagglutinin protein. Viral strains are indistinguishable serologically, although characteristic nucleotide sequences differentiate 24 genotypes. In this work, we describe a distant evolutionary branch within genotype D4. Designated subgenotype D4.2, this virus is distinguishable by neutralization with vaccine-induced monoclonal antibodies that target the neutralizing epitope (NE). The subgenotype D4.2 viruses have a higher predominance in countries with intermediary levels of vaccine coverage. Our studies demonstrate that subgenotype D4.2 lacks epitopes associated with half of the known antigenic sites, which significantly impacts our understanding of measles virus evolution. PMID:28356529
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Garamszegi, Sara; Franzosa, Eric A.; Xia, Yu
2013-01-01
A central challenge in host-pathogen systems biology is the elucidation of general, systems-level principles that distinguish host-pathogen interactions from within-host interactions. Current analyses of host-pathogen and within-host protein-protein interaction networks are largely limited by their resolution, treating proteins as nodes and interactions as edges. Here, we construct a domain-resolved map of human-virus and within-human protein-protein interaction networks by annotating protein interactions with high-coverage, high-accuracy, domain-centric interaction mechanisms: (1) domain-domain interactions, in which a domain in one protein binds to a domain in a second protein, and (2) domain-motif interactions, in which a domain in one protein binds to a short, linear peptide motif in a second protein. Analysis of these domain-resolved networks reveals, for the first time, significant mechanistic differences between virus-human and within-human interactions at the resolution of single domains. While human proteins tend to compete with each other for domain binding sites by means of sequence similarity, viral proteins tend to compete with human proteins for domain binding sites in the absence of sequence similarity. Independent of their previously established preference for targeting human protein hubs, viral proteins also preferentially target human proteins containing linear motif-binding domains. Compared to human proteins, viral proteins participate in more domain-motif interactions, target more unique linear motif-binding domains per residue, and contain more unique linear motifs per residue. Together, these results suggest that viruses surmount genome size constraints by convergently evolving multiple short linear motifs in order to effectively mimic, hijack, and manipulate complex host processes for their survival. Our domain-resolved analyses reveal unique signatures of pleiotropy, economy, and convergent evolution in viral-host interactions that are otherwise hidden in the traditional binary network, highlighting the power and necessity of high-resolution approaches in host-pathogen systems biology. PMID:24339775
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Austin, Christopher M; Hammer, Michael P; Lee, Yin Peng; Gan, Han Ming
2018-01-01
Abstract Background Some of the most widely recognized coral reef fishes are clownfish or anemonefish, members of the family Pomacentridae (subfamily: Amphiprioninae). They are popular aquarium species due to their bright colours, adaptability to captivity, and fascinating behavior. Their breeding biology (sequential hermaphrodites) and symbiotic mutualism with sea anemones have attracted much scientific interest. Moreover, there are some curious geographic-based phenotypes that warrant investigation. Leveraging on the advancement in Nanopore long read technology, we report the first hybrid assembly of the clown anemonefish (Amphiprion ocellaris) genome utilizing Illumina and Nanopore reads, further demonstrating the substantial impact of modest long read sequencing data sets on improving genome assembly statistics. Results We generated 43 Gb of short Illumina reads and 9 Gb of long Nanopore reads, representing approximate genome coverage of 54× and 11×, respectively, based on the range of estimated k-mer-predicted genome sizes of between 791 and 967 Mbp. The final assembled genome is contained in 6404 scaffolds with an accumulated length of 880 Mb (96.3% BUSCO-calculated genome completeness). Compared with the Illumina-only assembly, the hybrid approach generated 94% fewer scaffolds with an 18-fold increase in N50 length (401 kb) and increased the genome completeness by an additional 16%. A total of 27 240 high-quality protein-coding genes were predicted from the clown anemonefish, 26 211 (96%) of which were annotated functionally with information from either sequence homology or protein signature searches. Conclusions We present the first genome of any anemonefish and demonstrate the value of low coverage (∼11×) long Nanopore read sequencing in improving both genome assembly contiguity and completeness. The near-complete assembly of the A. ocellaris genome will be an invaluable molecular resource for supporting a range of genetic, genomic, and phylogenetic studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae. PMID:29342277
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Tan, Mun Hua; Austin, Christopher M; Hammer, Michael P; Lee, Yin Peng; Croft, Laurence J; Gan, Han Ming
2018-03-01
Some of the most widely recognized coral reef fishes are clownfish or anemonefish, members of the family Pomacentridae (subfamily: Amphiprioninae). They are popular aquarium species due to their bright colours, adaptability to captivity, and fascinating behavior. Their breeding biology (sequential hermaphrodites) and symbiotic mutualism with sea anemones have attracted much scientific interest. Moreover, there are some curious geographic-based phenotypes that warrant investigation. Leveraging on the advancement in Nanopore long read technology, we report the first hybrid assembly of the clown anemonefish (Amphiprion ocellaris) genome utilizing Illumina and Nanopore reads, further demonstrating the substantial impact of modest long read sequencing data sets on improving genome assembly statistics. We generated 43 Gb of short Illumina reads and 9 Gb of long Nanopore reads, representing approximate genome coverage of 54× and 11×, respectively, based on the range of estimated k-mer-predicted genome sizes of between 791 and 967 Mbp. The final assembled genome is contained in 6404 scaffolds with an accumulated length of 880 Mb (96.3% BUSCO-calculated genome completeness). Compared with the Illumina-only assembly, the hybrid approach generated 94% fewer scaffolds with an 18-fold increase in N50 length (401 kb) and increased the genome completeness by an additional 16%. A total of 27 240 high-quality protein-coding genes were predicted from the clown anemonefish, 26 211 (96%) of which were annotated functionally with information from either sequence homology or protein signature searches. We present the first genome of any anemonefish and demonstrate the value of low coverage (∼11×) long Nanopore read sequencing in improving both genome assembly contiguity and completeness. The near-complete assembly of the A. ocellaris genome will be an invaluable molecular resource for supporting a range of genetic, genomic, and phylogenetic studies specifically for clownfish and more generally for other related fish species of the family Pomacentridae.
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Nair, Shalima S; Luu, Phuc-Loi; Qu, Wenjia; Maddugoda, Madhavi; Huschtscha, Lily; Reddel, Roger; Chenevix-Trench, Georgia; Toso, Martina; Kench, James G; Horvath, Lisa G; Hayes, Vanessa M; Stricker, Phillip D; Hughes, Timothy P; White, Deborah L; Rasko, John E J; Wong, Justin J-L; Clark, Susan J
2018-05-28
Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing. In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×). Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.
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RefCNV: Identification of Gene-Based Copy Number Variants Using Whole Exome Sequencing.
Chang, Lun-Ching; Das, Biswajit; Lih, Chih-Jian; Si, Han; Camalier, Corinne E; McGregor, Paul M; Polley, Eric
2016-01-01
With rapid advances in DNA sequencing technologies, whole exome sequencing (WES) has become a popular approach for detecting somatic mutations in oncology studies. The initial intent of WES was to characterize single nucleotide variants, but it was observed that the number of sequencing reads that mapped to a genomic region correlated with the DNA copy number variants (CNVs). We propose a method RefCNV that uses a reference set to estimate the distribution of the coverage for each exon. The construction of the reference set includes an evaluation of the sources of variability in the coverage distribution. We observed that the processing steps had an impact on the coverage distribution. For each exon, we compared the observed coverage with the expected normal coverage. Thresholds for determining CNVs were selected to control the false-positive error rate. RefCNV prediction correlated significantly (r = 0.96-0.86) with CNV measured by digital polymerase chain reaction for MET (7q31), EGFR (7p12), or ERBB2 (17q12) in 13 tumor cell lines. The genome-wide CNV analysis showed a good overall correlation (Spearman's coefficient = 0.82) between RefCNV estimation and publicly available CNV data in Cancer Cell Line Encyclopedia. RefCNV also showed better performance than three other CNV estimation methods in genome-wide CNV analysis.
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Kuleshov, K V; Markelov, M L; Dedkov, V G; Vodop'ianov, A S; Kermanov, A V; Pisanov, R V; Kruglikov, V D; Mazrukho, A B; Maleev, V V; Shipulin, G A
2013-01-01
Determination of origin of 2 Vibrio cholerae strains isolated on the territory of Rostov region by using full genome sequencing data. Toxigenic strain 2011 EL- 301 V. cholerae 01 El Tor Inaba No. 301 (ctxAB+, tcpA+) and nontoxigenic strain V. cholerae O1 Ogawa P- 18785 (ctxAB-, tcpA+) were studied. Sequencing was carried out on the MiSeq platform. Phylogenetic analysis of the genomes obtained was carried out based on comparison of conservative part of the studied and 54 previously sequenced genomes. 2011EL-301 strain genome was presented by 164 contigs with an average coverage of 100, N50 parameter was 132 kb, for strain P- 18785 - 159 contigs with a coverage of69, N50 - 83 kb. The contigs obtained for strain 2011 EL-301 were deposited in DDBJ/EMBL/GenBank databases with access code AJFN02000000, for strain P-18785 - ANHS00000000. 716 protein-coding orthologous genes were detected. Based on phylogenetic analysis strain P- 18785 belongs to PG-1 subgroup (a group of predecessor strains of the 7th pandemic). Strain 2011EL-301 belongs to groups of strains of the 7th pandemic and is included into the cluster with later isolates that are associated with cases of cholera in South Africa and cases of import of cholera to the USA from Pakistan. The data obtained allows to establish phylogenetic connections with V cholerae strains isolated earlier.
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Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies.
Card, Daren C; Schield, Drew R; Reyes-Velasco, Jacobo; Fujita, Matthew K; Andrew, Audra L; Oyler-McCance, Sara J; Fike, Jennifer A; Tomback, Diana F; Ruggiero, Robert P; Castoe, Todd A
2014-01-01
As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5-5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.
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Two low coverage bird genomes and a comparison of reference-guided versus de novo genome assemblies
Card, Daren C.; Schield, Drew R.; Reyes-Velasco, Jacobo; Fujita, Matthre K.; Andrew, Audra L.; Oyler-McCance, Sara J.; Fike, Jennifer A.; Tomback, Diana F.; Ruggiero, Robert P.; Castoe, Todd A.
2014-01-01
As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (~3.5–5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies.
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Calibrating genomic and allelic coverage bias in single-cell sequencing.
Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher
2015-04-16
Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.
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Calibrating genomic and allelic coverage bias in single-cell sequencing
Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher
2016-01-01
Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913
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Dividing to unveil protein microheterogeneities: A Traveling Wave Ion Mobility study
Halgand, F.; Habchi, Johnny; Cravello, Laetitia; Martinho, Marlène; Guigliarelli, Bruno; Longhi, Sonia
2011-01-01
Over-expression of a protein in a foreign host is often the only route toward an exhaustive characterization especially when purification from the natural source(s) is hardly achievable. The key issue in these studies relies on quality control of the purified recombinant protein to precisely determining its identity as well as any undesirable micro-heterogeneities. While standard proteomics approaches preclude unbiased search for modifications, the optional technique of top down MSMS requires the use of highly accurate and highly resolved experiments to reveal subtle sequence modifications. In the present study, the top down MSMS approach combined with Traveling Wave Ion Mobility (TWIM) separation was evaluated for its ability to achieve high sequence coverage and to reveal subtle micro-heterogeneities that were hitherto only accessible with FTICR-MS instruments. The power of this approach is herein illustrated in an in-depth analysis of both wt and K496C variant of the recombinant X domain (XD, aa 459-507) of the measles virus phosphoprotein expressed in E. coli. Using top down MSMS combined to TWIM, we show that XD samples occasionally exhibit a micro-heterogeneity that could not be anticipated from the nucleotide sequence of the encoding constructs and that likely reflects a genetic drift, neutral or not, occurring during expression. In addition, an MTSL nitroxide probe that was grafted on the K496C XD variant was shown to undergo oxidation and/or protonation in the ESI source leading to artifactual mass increases. PMID:21800924
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Sexual differences in the sialomes of the zebra tick, Rhipicephalus pulchellus
Tan, Angelina W. L.; Francischetti, Ivo M.B.; Slovak, Mirko; Manjunatha, Kini R.; Ribeiro, José M. C.
2015-01-01
Ticks rely exclusively on vertebrate blood for their survival. During feeding ticks inject into their hosts a sophisticated salivary potion that overcomes host hemostasis and adverse inflammatory responses. These mediators may also enhance pathogen transmission. Knowledge of the tick salivary protein repertoire may lead to vaccine targets to disrupt feeding and/or parasite transmission as well as to the discovery of novel pharmacological agents. Male saliva may also assist reproduction because males use their mouthparts to lubricate and introduce their spermatophores into the females’ genital pore. The analyses of the sialomes of male and female ticks independently allow us to understand the strategy used by each gender to feed successfully.We sequenced cDNA libraries from pools of salivary glands from adult male and female R. pulchellus feeding at different time points, using the Illumina HiSeq protocol. De novo assembly of a total of 241,229,128 paired-end reads lead to extraction of 50,460 coding sequences (CDS), 11,277 of which had more than 75% coverage to known transcripts, or represented novel sequences, and were submitted to GenBank. Additionally, we generated the proteome, from the salivary gland extracts of male and female R. pulchellus, yielding a total of 454 and 2,063 proteins respectively which were identified by one or more peptides with at least 95% confidence. The data set is presented as an annotated hyperlinked Excel spreadsheet, describing 121 putative secreted protein families. Female and male specific transcripts were identified. PMID:25576852
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Marjanovic, Irena; Kostic, Jelena; Stanic, Bojana; Pejanovic, Nadja; Lucic, Bojana; Karan-Djurasevic, Teodora; Janic, Dragana; Dokmanovic, Lidija; Jankovic, Srdja; Vukovic, Nada Suvajdzic; Tomin, Dragica; Perisic, Ognjen; Rakocevic, Goran; Popovic, Milos; Pavlovic, Sonja; Tosic, Natasa
2016-10-01
The age-specific differences in the genetic mechanisms of myeloid leukemogenesis have been observed and studied previously. However, NGS technology has provided a possibility to obtain a large amount of mutation data. We analyzed DNA samples from 20 childhood (cAML) and 20 adult AML (aAML) patients, using NGS targeted sequencing. The average coverage of high-quality sequences was 2981 × per amplicon. A total of 412 (207 cAML, 205 aAML) variants in the coding regions were detected; out of which, only 122 (62 cAML and 60 aAML) were potentially protein-changing. Our results confirmed that AML contains small number of genetic alterations (median 3 mutations/patient in both groups). The prevalence of the most frequent single gene AML associated mutations differed in cAML and aAML patient cohorts: IDH1 (0 % cAML, 5 % aAML), IDH2 (0 % cAML, 10 % aAML), NPM1 (10 % cAML, 35 % aAML). Additionally, potentially protein-changing variants were found in tyrosine kinase genes or genes encoding tyrosine kinase associated proteins (JAK3, ABL1, GNAQ, and EGFR) in cAML, while among aAML, the prevalence is directed towards variants in the methylation and histone modifying genes (IDH1, IDH2, and SMARCB1). Besides uniform genomic profile of AML, specific genetic characteristic was exclusively detected in cAML and aAML.
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Using Tablet for visual exploration of second-generation sequencing data.
Milne, Iain; Stephen, Gordon; Bayer, Micha; Cock, Peter J A; Pritchard, Leighton; Cardle, Linda; Shaw, Paul D; Marshall, David
2013-03-01
The advent of second-generation sequencing (2GS) has provided a range of significant new challenges for the visualization of sequence assemblies. These include the large volume of data being generated, short-read lengths and different data types and data formats associated with the diversity of new sequencing technologies. This article illustrates how Tablet-a high-performance graphical viewer for visualization of 2GS assemblies and read mappings-plays an important role in the analysis of these data. We present Tablet, and through a selection of use cases, demonstrate its value in quality assurance and scientific discovery, through features such as whole-reference coverage overviews, variant highlighting, paired-end read mark-up, GFF3-based feature tracks and protein translations. We discuss the computing and visualization techniques utilized to provide a rich and responsive graphical environment that enables users to view a range of file formats with ease. Tablet installers can be freely downloaded from http://bioinf.hutton.ac.uk/tablet in 32 or 64-bit versions for Windows, OS X, Linux or Solaris. For further details on the Tablet, contact tablet@hutton.ac.uk.
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The genome of woodland strawberry (Fragaria vesca)
Shulaev, Vladimir; Sargent, Daniel J; Crowhurst, Ross N; Mockler, Todd C; Folkerts, Otto; Delcher, Arthur L; Jaiswal, Pankaj; Mockaitis, Keithanne; Liston, Aaron; Mane, Shrinivasrao P; Burns, Paul; Davis, Thomas M; Slovin, Janet P; Bassil, Nahla; Hellens, Roger P; Evans, Clive; Harkins, Tim; Kodira, Chinnappa; Desany, Brian; Crasta, Oswald R; Jensen, Roderick V; Allan, Andrew C; Michael, Todd P; Setubal, Joao Carlos; Celton, Jean-Marc; Rees, D Jasper G; Williams, Kelly P; Holt, Sarah H; Ruiz Rojas, Juan Jairo; Chatterjee, Mithu; Liu, Bo; Silva, Herman; Meisel, Lee; Adato, Avital; Filichkin, Sergei A; Troggio, Michela; Viola, Roberto; Ashman, Tia-Lynn; Wang, Hao; Dharmawardhana, Palitha; Elser, Justin; Raja, Rajani; Priest, Henry D; Bryant, Douglas W; Fox, Samuel E; Givan, Scott A; Wilhelm, Larry J; Naithani, Sushma; Christoffels, Alan; Salama, David Y; Carter, Jade; Girona, Elena Lopez; Zdepski, Anna; Wang, Wenqin; Kerstetter, Randall A; Schwab, Wilfried; Korban, Schuyler S; Davik, Jahn; Monfort, Amparo; Denoyes-Rothan, Beatrice; Arus, Pere; Mittler, Ron; Flinn, Barry; Aharoni, Asaph; Bennetzen, Jeffrey L; Salzberg, Steven L; Dickerman, Allan W; Velasco, Riccardo; Borodovsky, Mark; Veilleux, Richard E; Folta, Kevin M
2012-01-01
The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted. PMID:21186353
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Recurrent hyperactive ESR1 fusion proteins in endocrine therapy-resistant breast cancer.
Hartmaier, R J; Trabucco, S E; Priedigkeit, N; Chung, J H; Parachoniak, C A; Vanden Borre, P; Morley, S; Rosenzweig, M; Gay, L M; Goldberg, M E; Suh, J; Ali, S M; Ross, J; Leyland-Jones, B; Young, B; Williams, C; Park, B; Tsai, M; Haley, B; Peguero, J; Callahan, R D; Sachelarie, I; Cho, J; Atkinson, J M; Bahreini, A; Nagle, A M; Puhalla, S L; Watters, R J; Erdogan-Yildirim, Z; Cao, L; Oesterreich, S; Mathew, A; Lucas, P C; Davidson, N E; Brufsky, A M; Frampton, G M; Stephens, P J; Chmielecki, J; Lee, A V
2018-04-01
Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287-395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3' partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6-7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.
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Modeling the SHG activities of diverse protein crystals
DOE Office of Scientific and Technical Information (OSTI.GOV)
Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J., E-mail: gsimpson@purdue.edu
2012-11-01
The origins of the diversity in the SHG signal from protein crystals are investigated and potential protein-crystal coverage by SHG microscopy is assessed. A symmetry-additive ab initio model for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much ofmore » the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within the crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ∼84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.« less
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Tabata, Ryo; Kamiya, Takehiro; Shigenobu, Shuji; Yamaguchi, Katsushi; Yamada, Masashi; Hasebe, Mitsuyasu; Fujiwara, Toru; Sawa, Shinichiro
2013-01-01
Next-generation sequencing (NGS) technologies enable the rapid production of an enormous quantity of sequence data. These powerful new technologies allow the identification of mutations by whole-genome sequencing. However, most reported NGS-based mapping methods, which are based on bulked segregant analysis, are costly and laborious. To address these limitations, we designed a versatile NGS-based mapping method that consists of a combination of low- to medium-coverage multiplex SOLiD (Sequencing by Oligonucleotide Ligation and Detection) and classical genetic rough mapping. Using only low to medium coverage reduces the SOLiD sequencing costs and, since just 10 to 20 mutant F2 plants are required for rough mapping, the operation is simple enough to handle in a laboratory with limited space and funding. As a proof of principle, we successfully applied this method to identify the CTR1, which is involved in boron-mediated root development, from among a population of high boron requiring Arabidopsis thaliana mutants. Our work demonstrates that this NGS-based mapping method is a moderately priced and versatile method that can readily be applied to other model organisms. PMID:23104114
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2013-01-01
Background The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Results Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. Conclusions In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data. PMID:23442253
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Ye, Hongping; Hill, John; Kauffman, John; Gryniewicz, Connie; Han, Xianlin
2013-01-01
iTRAQ (isotope tags for relative and absolute quantification) reagent coupled with MALDI TOF/TOF mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies, and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, Lantus®) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including Human insulin (P28K29) and Lispro (K28P29), which only differ by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting. PMID:18489896
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Li, Ran; Zhang, Meng-Yi; Liu, Yu-Wei; Zhang, Zheng; Smagghe, Guy; Wang, Jin-Jun
2017-01-01
Time-dependent expression of proteins in ovary is important to understand oogenesis in insects. Here, we profiled the proteomes of developing ovaries from Bactrocera dorsalis (Hendel) to obtain information about ovarian development with particular emphasis on differentially expressed proteins (DEPs) involved in oogenesis. A total of 4838 proteins were identified with an average peptide number of 8.15 and sequence coverage of 20.79%. Quantitative proteomic analysis showed that a total of 612 and 196 proteins were differentially expressed in developing and mature ovaries, respectively. Furthermore, 153, 196 and 59 potential target proteins were highly expressed in early, vitellogenic and mature ovaries and most tested DEPs had the similar trends consistent with the respective transcriptional profiles. These proteins were abundantly expressed in pre-vitellogenic and vitellogenic stages, including tropomyosin, vitellogenin, eukaryotic translation initiation factor, heat shock protein, importin protein, vitelline membrane protein, and chorion protein. Several hormone and signal pathway related proteins were also identified during ovarian development including piRNA, notch, insulin, juvenile, and ecdysone hormone signal pathways. This is the first report of a global ovary proteome of a tephritid fruit fly, and may contribute to understanding the complicate processes of ovarian development and exploring the potentially novel pest control targets. PMID:28665301
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Analysis of quality raw data of second generation sequencers with Quality Assessment Software.
Ramos, Rommel Tj; Carneiro, Adriana R; Baumbach, Jan; Azevedo, Vasco; Schneider, Maria Pc; Silva, Artur
2011-04-18
Second generation technologies have advantages over Sanger; however, they have resulted in new challenges for the genome construction process, especially because of the small size of the reads, despite the high degree of coverage. Independent of the program chosen for the construction process, DNA sequences are superimposed, based on identity, to extend the reads, generating contigs; mismatches indicate a lack of homology and are not included. This process improves our confidence in the sequences that are generated. We developed Quality Assessment Software, with which one can review graphs showing the distribution of quality values from the sequencing reads. This software allow us to adopt more stringent quality standards for sequence data, based on quality-graph analysis and estimated coverage after applying the quality filter, providing acceptable sequence coverage for genome construction from short reads. Quality filtering is a fundamental step in the process of constructing genomes, as it reduces the frequency of incorrect alignments that are caused by measuring errors, which can occur during the construction process due to the size of the reads, provoking misassemblies. Application of quality filters to sequence data, using the software Quality Assessment, along with graphing analyses, provided greater precision in the definition of cutoff parameters, which increased the accuracy of genome construction.
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Croft, Nathan P.; de Verteuil, Danielle A.; Smith, Stewart A.; Wong, Yik Chun; Schittenhelm, Ralf B.; Tscharke, David C.; Purcell, Anthony W.
2015-01-01
The generation of antigen-specific reagents is a significant bottleneck in the study of complex pathogens that express many hundreds to thousands of different proteins or to emerging or new strains of viruses that display potential pandemic qualities and therefore require rapid investigation. In these instances the development of antibodies for example can be prohibitively expensive to cover the full pathogen proteome, or the lead time may be unacceptably long in urgent cases where new highly pathogenic viral strains may emerge. Because genomic information on such pathogens can be rapidly acquired this opens up avenues using mass spectrometric approaches to study pathogen antigen expression, host responses and for screening the utility of therapeutics. In particular, data-independent acquisition (DIA) modalities on high-resolution mass spectrometers generate spectral information on all components of a complex sample providing depth of coverage hitherto only seen in genomic deep sequencing. The spectral information generated by DIA can be iteratively interrogated for potentially any protein of interest providing both evidence of protein expression and quantitation. Here we apply a solely DIA mass spectrometry based methodology to profile the viral antigen expression in cells infected with vaccinia virus up to 9 h post infection without the need for antigen specific antibodies or other reagents. We demonstrate deep coverage of the vaccinia virus proteome using a SWATH-MS acquisition approach, extracting quantitative kinetics of 100 virus proteins within a single experiment. The results highlight the complexity of vaccinia protein expression, complementing what is known at the transcriptomic level, and provide a valuable resource and technique for future studies of viral infection and replication kinetics. Furthermore, they highlight the utility of DIA and mass spectrometry in the dissection of host-pathogen interactions. PMID:25755296
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High precision multi-genome scale reannotation of enzyme function by EFICAz
Arakaki, Adrian K; Tian, Weidong; Skolnick, Jeffrey
2006-01-01
Background The functional annotation of most genes in newly sequenced genomes is inferred from similarity to previously characterized sequences, an annotation strategy that often leads to erroneous assignments. We have performed a reannotation of 245 genomes using an updated version of EFICAz, a highly precise method for enzyme function prediction. Results Based on our three-field EC number predictions, we have obtained lower-bound estimates for the average enzyme content in Archaea (29%), Bacteria (30%) and Eukarya (18%). Most annotations added in KEGG from 2005 to 2006 agree with EFICAz predictions made in 2005. The coverage of EFICAz predictions is significantly higher than that of KEGG, especially for eukaryotes. Thousands of our novel predictions correspond to hypothetical proteins. We have identified a subset of 64 hypothetical proteins with low sequence identity to EFICAz training enzymes, whose biochemical functions have been recently characterized and find that in 96% (84%) of the cases we correctly identified their three-field (four-field) EC numbers. For two of the 64 hypothetical proteins: PA1167 from Pseudomonas aeruginosa, an alginate lyase (EC 4.2.2.3) and Rv1700 of Mycobacterium tuberculosis H37Rv, an ADP-ribose diphosphatase (EC 3.6.1.13), we have detected annotation lag of more than two years in databases. Two examples are presented where EFICAz predictions act as hypothesis generators for understanding the functional roles of hypothetical proteins: FLJ11151, a human protein overexpressed in cancer that EFICAz identifies as an endopolyphosphatase (EC 3.6.1.10), and MW0119, a protein of Staphylococcus aureus strain MW2 that we propose as candidate virulence factor based on its EFICAz predicted activity, sphingomyelin phosphodiesterase (EC 3.1.4.12). Conclusion Our results suggest that we have generated enzyme function annotations of high precision and recall. These predictions can be mined and correlated with other information sources to generate biologically significant hypotheses and can be useful for comparative genome analysis and automated metabolic pathway reconstruction. PMID:17166279
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Stigter, E C A; de Jong, G J; van Bennekom, W P
2008-07-07
On-line digestion of proteins under acidic conditions was studied using micro-reactors consisting of dextran-modified fused-silica capillaries with covalently immobilized pepsin. The proteins used in this study differed in molecular weight, isoelectric point and sample composition. The injected protein samples were completely digested in 3 min and the digest was analyzed with micro-high performance liquid chromatography (HPLC) and tandem mass spectrometry (MS/MS). The different proteins present in the samples could be identified with a Mascot database search on the basis of auto-MS/MS data. It proved also to be possible to digest and analyze protein mixtures with a sequence coverage of 55% and 97% for the haemoglobin beta- and alpha-chain, respectively, and 35-55% for the various casein variants. Protease auto-digestion, sample carry-over and loss of signal due to adsorption of the injected proteins were not observed. The backpressure of the reactor is low which makes coupling to systems such as Surface Plasmon Resonance biosensors, which do not tolerate too high pressure, possible. The reactor was stable for at least 40 days when used continuously.
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Omelchenko, Marina V; Galperin, Michael Y; Wolf, Yuri I; Koonin, Eugene V
2010-04-30
Evolutionarily unrelated proteins that catalyze the same biochemical reactions are often referred to as analogous - as opposed to homologous - enzymes. The existence of numerous alternative, non-homologous enzyme isoforms presents an interesting evolutionary problem; it also complicates genome-based reconstruction of the metabolic pathways in a variety of organisms. In 1998, a systematic search for analogous enzymes resulted in the identification of 105 Enzyme Commission (EC) numbers that included two or more proteins without detectable sequence similarity to each other, including 34 EC nodes where proteins were known (or predicted) to have distinct structural folds, indicating independent evolutionary origins. In the past 12 years, many putative non-homologous isofunctional enzymes were identified in newly sequenced genomes. In addition, efforts in structural genomics resulted in a vastly improved structural coverage of proteomes, providing for definitive assessment of (non)homologous relationships between proteins. We report the results of a comprehensive search for non-homologous isofunctional enzymes (NISE) that yielded 185 EC nodes with two or more experimentally characterized - or predicted - structurally unrelated proteins. Of these NISE sets, only 74 were from the original 1998 list. Structural assignments of the NISE show over-representation of proteins with the TIM barrel fold and the nucleotide-binding Rossmann fold. From the functional perspective, the set of NISE is enriched in hydrolases, particularly carbohydrate hydrolases, and in enzymes involved in defense against oxidative stress. These results indicate that at least some of the non-homologous isofunctional enzymes were recruited relatively recently from enzyme families that are active against related substrates and are sufficiently flexible to accommodate changes in substrate specificity.
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The protein structure prediction problem could be solved using the current PDB library
Zhang, Yang; Skolnick, Jeffrey
2005-01-01
For single-domain proteins, we examine the completeness of the structures in the current Protein Data Bank (PDB) library for use in full-length model construction of unknown sequences. To address this issue, we employ a comprehensive benchmark set of 1,489 medium-size proteins that cover the PDB at the level of 35% sequence identity and identify templates by structure alignment. With homologous proteins excluded, we can always find similar folds to native with an average rms deviation (RMSD) from native of 2.5 Å with ≈82% alignment coverage. These template structures often contain a significant number of insertions/deletions. The tasser algorithm was applied to build full-length models, where continuous fragments are excised from the top-scoring templates and reassembled under the guide of an optimized force field, which includes consensus restraints taken from the templates and knowledge-based statistical potentials. For almost all targets (except for 2/1,489), the resultant full-length models have an RMSD to native below 6 Å (97% of them below 4 Å). On average, the RMSD of full-length models is 2.25 Å, with aligned regions improved from 2.5 Å to 1.88 Å, comparable with the accuracy of low-resolution experimental structures. Furthermore, starting from state-of-the-art structural alignments, we demonstrate a methodology that can consistently bring template-based alignments closer to native. These results are highly suggestive that the protein-folding problem can in principle be solved based on the current PDB library by developing efficient fold recognition algorithms that can recover such initial alignments. PMID:15653774
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MCMC-ODPR: primer design optimization using Markov Chain Monte Carlo sampling.
Kitchen, James L; Moore, Jonathan D; Palmer, Sarah A; Allaby, Robin G
2012-11-05
Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base.
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MCMC-ODPR: Primer design optimization using Markov Chain Monte Carlo sampling
2012-01-01
Background Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. Results After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. Conclusions MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base. PMID:23126469
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Hackett, Justin B; Lu, Yan
2017-05-04
In land plants, plastid and mitochondrial RNAs are subject to post-transcriptional C-to-U RNA editing. T-DNA insertions in the ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 gene resulted in reduced photosystem II (PSII) activity and smaller plant and leaf sizes. Exon coverage analysis of the ORRM6 gene showed that orrm6-1 and orrm6-2 are loss-of-function mutants. Compared to other ORRM proteins, ORRM6 affects a relative small number of RNA editing sites. Sanger sequencing of reverse transcription-PCR products of plastid transcripts revealed 2 plastid RNA editing sites that are substantially affected in the orrm6 mutants: psbF-C77 and accD-C794. The psbF gene encodes the β subunit of cytochrome b 559 , an essential component of PSII. The accD gene encodes the β subunit of acetyl-CoA carboxylase, a protein required in plastid fatty acid biosynthesis. Whole-transcriptome RNA-seq demonstrated that editing at psbF-C77 is nearly absent and the editing extent at accD-C794 was significantly reduced. Gene set enrichment pathway analysis showed that expression of multiple gene sets involved in photosynthesis, especially photosynthetic electron transport, is significantly upregulated in both orrm6 mutants. The upregulation could be a mechanism to compensate for the reduced PSII electron transport rate in the orrm6 mutants. These results further demonstrated that Organelle RNA Recognition Motif protein ORRM6 is required in editing of specific RNAs in the Arabidopsis (Arabidopsis thaliana) plastid.
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Development of a Model System for Tick-Borne Flavivirus Persistence in HEK 293T Cells
Mlera, Luwanika; Offerdahl, Danielle K.; Martens, Craig; Porcella, Stephen F.; Melik, Wessam
2015-01-01
ABSTRACT We devised a model system to study persistent infection by the tick-borne flavivirus Langat virus (LGTV) in 293T cells. Infection with a molecularly cloned LGTV strain produced an acute lytic crisis that left few surviving cells. The culture was repopulated by cells that were ~90% positive for LGTV E protein, thus initiating a persistent infection that was maintained for at least 35 weeks without additional lytic crises. Staining of cells for viral proteins and ultrastructural analysis revealed only minor differences from the acute phase of infection. Infectious LGTV decreased markedly over the study period, but the number of viral genomes remained relatively constant, suggesting the development of defective interfering particles (DIPs). Viral genome changes were investigated by RNA deep sequencing. At the initiation of persistent infection, levels of DIPs were below the limit of detection at a coverage depth of 11,288-fold, implying that DIPs are not required for initiation of persistence. However, after 15 passages, DIPs constituted approximately 34% of the total LGTV population (coverage of 1,293-fold). Furthermore, at this point, one specific DIP population predominated in which nucleotides 1058 to 2881 had been deleted. This defective genome specified an intact polyprotein that coded for a truncated fusion protein containing 28 N-terminal residues of E and 134 C-terminal residues of NS1. Such a fusion protein has not previously been described, and a possible function in persistent infection is uncertain. DIPs are not required for the initiation of persistent LGTV infection but may play a role in the maintenance of viral persistence. PMID:26045539
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Mapping the protein domain structures of the respiratory mucins: a mucin proteome coverage study.
Cao, Rui; Wang, T Tiffany; DeMaria, Genevieve; Sheehan, John K; Kesimer, Mehmet
2012-08-03
Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides that greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion, and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel-forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from 10 different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or "spontaneous cleavages". Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20, which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.
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Identification of copy number variants in whole-genome data using Reference Coverage Profiles
Glusman, Gustavo; Severson, Alissa; Dhankani, Varsha; Robinson, Max; Farrah, Terry; Mauldin, Denise E.; Stittrich, Anna B.; Ament, Seth A.; Roach, Jared C.; Brunkow, Mary E.; Bodian, Dale L.; Vockley, Joseph G.; Shmulevich, Ilya; Niederhuber, John E.; Hood, Leroy
2015-01-01
The identification of DNA copy numbers from short-read sequencing data remains a challenge for both technical and algorithmic reasons. The raw data for these analyses are measured in tens to hundreds of gigabytes per genome; transmitting, storing, and analyzing such large files is cumbersome, particularly for methods that analyze several samples simultaneously. We developed a very efficient representation of depth of coverage (150–1000× compression) that enables such analyses. Current methods for analyzing variants in whole-genome sequencing (WGS) data frequently miss copy number variants (CNVs), particularly hemizygous deletions in the 1–100 kb range. To fill this gap, we developed a method to identify CNVs in individual genomes, based on comparison to joint profiles pre-computed from a large set of genomes. We analyzed depth of coverage in over 6000 high quality (>40×) genomes. The depth of coverage has strong sequence-specific fluctuations only partially explained by global parameters like %GC. To account for these fluctuations, we constructed multi-genome profiles representing the observed or inferred diploid depth of coverage at each position along the genome. These Reference Coverage Profiles (RCPs) take into account the diverse technologies and pipeline versions used. Normalization of the scaled coverage to the RCP followed by hidden Markov model (HMM) segmentation enables efficient detection of CNVs and large deletions in individual genomes. Use of pre-computed multi-genome coverage profiles improves our ability to analyze each individual genome. We make available RCPs and tools for performing these analyses on personal genomes. We expect the increased sensitivity and specificity for individual genome analysis to be critical for achieving clinical-grade genome interpretation. PMID:25741365
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Giannone, Richard J.; Wurch, Louie L.; Podar, Mircea
The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. It is thought that this interaction is membrane-associated, involving a myriad of membrane-anchored proteins; proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitansproteins. We show that proteins with increased hydrophobic character, includingmore » membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. Moreover, these gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.« less
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Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum
DOE Office of Scientific and Technical Information (OSTI.GOV)
VanBuren, Robert; Bryant, Doug; Edger, Patrick P.
Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less
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Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum
VanBuren, Robert; Bryant, Doug; Edger, Patrick P.; ...
2015-11-11
Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly1. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetiummore » genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. As a result, the Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.« less
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De novo peptide sequencing by deep learning
Tran, Ngoc Hieu; Zhang, Xianglilan; Xin, Lei; Shan, Baozhen; Li, Ming
2017-01-01
De novo peptide sequencing from tandem MS data is the key technology in proteomics for the characterization of proteins, especially for new sequences, such as mAbs. In this study, we propose a deep neural network model, DeepNovo, for de novo peptide sequencing. DeepNovo architecture combines recent advances in convolutional neural networks and recurrent neural networks to learn features of tandem mass spectra, fragment ions, and sequence patterns of peptides. The networks are further integrated with local dynamic programming to solve the complex optimization task of de novo sequencing. We evaluated the method on a wide variety of species and found that DeepNovo considerably outperformed state of the art methods, achieving 7.7–22.9% higher accuracy at the amino acid level and 38.1–64.0% higher accuracy at the peptide level. We further used DeepNovo to automatically reconstruct the complete sequences of antibody light and heavy chains of mouse, achieving 97.5–100% coverage and 97.2–99.5% accuracy, without assisting databases. Moreover, DeepNovo is retrainable to adapt to any sources of data and provides a complete end-to-end training and prediction solution to the de novo sequencing problem. Not only does our study extend the deep learning revolution to a new field, but it also shows an innovative approach in solving optimization problems by using deep learning and dynamic programming. PMID:28720701
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MSeq-CNV: accurate detection of Copy Number Variation from Sequencing of Multiple samples.
Malekpour, Seyed Amir; Pezeshk, Hamid; Sadeghi, Mehdi
2018-03-05
Currently a few tools are capable of detecting genome-wide Copy Number Variations (CNVs) based on sequencing of multiple samples. Although aberrations in mate pair insertion sizes provide additional hints for the CNV detection based on multiple samples, the majority of the current tools rely only on the depth of coverage. Here, we propose a new algorithm (MSeq-CNV) which allows detecting common CNVs across multiple samples. MSeq-CNV applies a mixture density for modeling aberrations in depth of coverage and abnormalities in the mate pair insertion sizes. Each component in this mixture density applies a Binomial distribution for modeling the number of mate pairs with aberration in the insertion size and also a Poisson distribution for emitting the read counts, in each genomic position. MSeq-CNV is applied on simulated data and also on real data of six HapMap individuals with high-coverage sequencing, in 1000 Genomes Project. These individuals include a CEU trio of European ancestry and a YRI trio of Nigerian ethnicity. Ancestry of these individuals is studied by clustering the identified CNVs. MSeq-CNV is also applied for detecting CNVs in two samples with low-coverage sequencing in 1000 Genomes Project and six samples form the Simons Genome Diversity Project.
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The impact of physiological crowding on the diffusivity of membrane bound proteins.
Houser, Justin R; Busch, David J; Bell, David R; Li, Brian; Ren, Pengyu; Stachowiak, Jeanne C
2016-02-21
Diffusion of transmembrane and peripheral membrane-bound proteins within the crowded cellular membrane environment is essential to diverse biological processes including cellular signaling, endocytosis, and motility. Nonetheless we presently lack a detailed understanding of the influence of physiological levels of crowding on membrane protein diffusion. Utilizing quantitative in vitro measurements, here we demonstrate that the diffusivities of membrane bound proteins follow a single linearly decreasing trend with increasing membrane coverage by proteins. This trend holds for homogenous protein populations across a range of protein sizes and for heterogeneous mixtures of proteins of different sizes, such that protein diffusivity is controlled by the total coverage of the surrounding membrane. These results demonstrate that steric exclusion within the crowded membrane environment can fundamentally limit the diffusive rate of proteins, regardless of their size. In cells this "speed limit" could be modulated by changes in local membrane coverage, providing a mechanism for tuning the rate of molecular interaction and assembly.
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Genome Wide Characterization of Simple Sequence Repeats in Cucumber
USDA-ARS?s Scientific Manuscript database
The whole genome sequence of the cucumber cultivar Gy14 was recently sequenced at 15× coverage with the Roche 454 Titanium technology. The microsatellite DNA sequences (simple sequence repeats, SSRs) in the assembled scaffolds were computationally explored and characterized. A total of 112,073 SSRs ...
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Two Low Coverage Bird Genomes and a Comparison of Reference-Guided versus De Novo Genome Assemblies
Card, Daren C.; Schield, Drew R.; Reyes-Velasco, Jacobo; Fujita, Matthew K.; Andrew, Audra L.; Oyler-McCance, Sara J.; Fike, Jennifer A.; Tomback, Diana F.; Ruggiero, Robert P.; Castoe, Todd A.
2014-01-01
As a greater number and diversity of high-quality vertebrate reference genomes become available, it is increasingly feasible to use these references to guide new draft assemblies for related species. Reference-guided assembly approaches may substantially increase the contiguity and completeness of a new genome using only low levels of genome coverage that might otherwise be insufficient for de novo genome assembly. We used low-coverage (∼3.5–5.5x) Illumina paired-end sequencing to assemble draft genomes of two bird species (the Gunnison Sage-Grouse, Centrocercus minimus, and the Clark's Nutcracker, Nucifraga columbiana). We used these data to estimate de novo genome assemblies and reference-guided assemblies, and compared the information content and completeness of these assemblies by comparing CEGMA gene set representation, repeat element content, simple sequence repeat content, and GC isochore structure among assemblies. Our results demonstrate that even lower-coverage genome sequencing projects are capable of producing informative and useful genomic resources, particularly through the use of reference-guided assemblies. PMID:25192061
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CNNdel: Calling Structural Variations on Low Coverage Data Based on Convolutional Neural Networks
2017-01-01
Many structural variations (SVs) detection methods have been proposed due to the popularization of next-generation sequencing (NGS). These SV calling methods use different SV-property-dependent features; however, they all suffer from poor accuracy when running on low coverage sequences. The union of results from these tools achieves fairly high sensitivity but still produces low accuracy on low coverage sequence data. That is, these methods contain many false positives. In this paper, we present CNNdel, an approach for calling deletions from paired-end reads. CNNdel gathers SV candidates reported by multiple tools and then extracts features from aligned BAM files at the positions of candidates. With labeled feature-expressed candidates as a training set, CNNdel trains convolutional neural networks (CNNs) to distinguish true unlabeled candidates from false ones. Results show that CNNdel works well with NGS reads from 26 low coverage genomes of the 1000 Genomes Project. The paper demonstrates that convolutional neural networks can automatically assign the priority of SV features and reduce the false positives efficaciously. PMID:28630866
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Kempton, Colton E.; Heninger, Justin R.; Johnson, Steven M.
2014-01-01
Nucleosomes and their positions in the eukaryotic genome play an important role in regulating gene expression by influencing accessibility to DNA. Many factors influence a nucleosome's final position in the chromatin landscape including the underlying genomic sequence. One of the primary reasons for performing in vitro nucleosome reconstitution experiments is to identify how the underlying DNA sequence will influence a nucleosome's position in the absence of other compounding cellular factors. However, concerns have been raised about the reproducibility of data generated from these kinds of experiments. Here we present data for in vitro nucleosome reconstitution experiments performed on linear plasmid DNA that demonstrate that, when coverage is deep enough, these reconstitution experiments are exquisitely reproducible and highly consistent. Our data also suggests that a coverage depth of 35X be maintained for maximal confidence when assaying nucleosome positions, but lower coverage levels may be generally sufficient. These coverage depth recommendations are sufficient in the experimental system and conditions used in this study, but may vary depending on the exact parameters used in other systems. PMID:25093869
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Bilton, Timothy P.; Schofield, Matthew R.; Black, Michael A.; Chagné, David; Wilcox, Phillip L.; Dodds, Ken G.
2018-01-01
Next-generation sequencing is an efficient method that allows for substantially more markers than previous technologies, providing opportunities for building high-density genetic linkage maps, which facilitate the development of nonmodel species’ genomic assemblies and the investigation of their genes. However, constructing genetic maps using data generated via high-throughput sequencing technology (e.g., genotyping-by-sequencing) is complicated by the presence of sequencing errors and genotyping errors resulting from missing parental alleles due to low sequencing depth. If unaccounted for, these errors lead to inflated genetic maps. In addition, map construction in many species is performed using full-sibling family populations derived from the outcrossing of two individuals, where unknown parental phase and varying segregation types further complicate construction. We present a new methodology for modeling low coverage sequencing data in the construction of genetic linkage maps using full-sibling populations of diploid species, implemented in a package called GUSMap. Our model is based on the Lander–Green hidden Markov model but extended to account for errors present in sequencing data. We were able to obtain accurate estimates of the recombination fractions and overall map distance using GUSMap, while most existing mapping packages produced inflated genetic maps in the presence of errors. Our results demonstrate the feasibility of using low coverage sequencing data to produce genetic maps without requiring extensive filtering of potentially erroneous genotypes, provided that the associated errors are correctly accounted for in the model. PMID:29487138
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Bilton, Timothy P; Schofield, Matthew R; Black, Michael A; Chagné, David; Wilcox, Phillip L; Dodds, Ken G
2018-05-01
Next-generation sequencing is an efficient method that allows for substantially more markers than previous technologies, providing opportunities for building high-density genetic linkage maps, which facilitate the development of nonmodel species' genomic assemblies and the investigation of their genes. However, constructing genetic maps using data generated via high-throughput sequencing technology ( e.g. , genotyping-by-sequencing) is complicated by the presence of sequencing errors and genotyping errors resulting from missing parental alleles due to low sequencing depth. If unaccounted for, these errors lead to inflated genetic maps. In addition, map construction in many species is performed using full-sibling family populations derived from the outcrossing of two individuals, where unknown parental phase and varying segregation types further complicate construction. We present a new methodology for modeling low coverage sequencing data in the construction of genetic linkage maps using full-sibling populations of diploid species, implemented in a package called GUSMap. Our model is based on the Lander-Green hidden Markov model but extended to account for errors present in sequencing data. We were able to obtain accurate estimates of the recombination fractions and overall map distance using GUSMap, while most existing mapping packages produced inflated genetic maps in the presence of errors. Our results demonstrate the feasibility of using low coverage sequencing data to produce genetic maps without requiring extensive filtering of potentially erroneous genotypes, provided that the associated errors are correctly accounted for in the model. Copyright © 2018 Bilton et al.
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firestar--advances in the prediction of functionally important residues.
Lopez, Gonzalo; Maietta, Paolo; Rodriguez, Jose Manuel; Valencia, Alfonso; Tress, Michael L
2011-07-01
firestar is a server for predicting catalytic and ligand-binding residues in protein sequences. Here, we present the important developments since the first release of firestar. Previous versions of the server required human interpretation of the results; the server is now fully automatized. firestar has been implemented as a web service and can now be run in high-throughput mode. Prediction coverage has been greatly improved with the extension of the FireDB database and the addition of alignments generated by HHsearch. Ligands in FireDB are now classified for biological relevance. Many of the changes have been motivated by the critical assessment of techniques for protein structure prediction (CASP) ligand-binding prediction experiment, which provided us with a framework to test the performance of firestar. URL: http://firedb.bioinfo.cnio.es/Php/FireStar.php.
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firestar—advances in the prediction of functionally important residues
Lopez, Gonzalo; Maietta, Paolo; Rodriguez, Jose Manuel; Valencia, Alfonso; Tress, Michael L.
2011-01-01
firestar is a server for predicting catalytic and ligand-binding residues in protein sequences. Here, we present the important developments since the first release of firestar. Previous versions of the server required human interpretation of the results; the server is now fully automatized. firestar has been implemented as a web service and can now be run in high-throughput mode. Prediction coverage has been greatly improved with the extension of the FireDB database and the addition of alignments generated by HHsearch. Ligands in FireDB are now classified for biological relevance. Many of the changes have been motivated by the critical assessment of techniques for protein structure prediction (CASP) ligand-binding prediction experiment, which provided us with a framework to test the performance of firestar. URL: http://firedb.bioinfo.cnio.es/Php/FireStar.php. PMID:21672959
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Low-Cost, High-Throughput Sequencing of DNA Assemblies Using a Highly Multiplexed Nextera Process.
Shapland, Elaine B; Holmes, Victor; Reeves, Christopher D; Sorokin, Elena; Durot, Maxime; Platt, Darren; Allen, Christopher; Dean, Jed; Serber, Zach; Newman, Jack; Chandran, Sunil
2015-07-17
In recent years, next-generation sequencing (NGS) technology has greatly reduced the cost of sequencing whole genomes, whereas the cost of sequence verification of plasmids via Sanger sequencing has remained high. Consequently, industrial-scale strain engineers either limit the number of designs or take short cuts in quality control. Here, we show that over 4000 plasmids can be completely sequenced in one Illumina MiSeq run for less than $3 each (15× coverage), which is a 20-fold reduction over using Sanger sequencing (2× coverage). We reduced the volume of the Nextera tagmentation reaction by 100-fold and developed an automated workflow to prepare thousands of samples for sequencing. We also developed software to track the samples and associated sequence data and to rapidly identify correctly assembled constructs having the fewest defects. As DNA synthesis and assembly become a centralized commodity, this NGS quality control (QC) process will be essential to groups operating high-throughput pipelines for DNA construction.
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The diploid genome sequence of an Asian individual
Wang, Jun; Wang, Wei; Li, Ruiqiang; Li, Yingrui; Tian, Geng; Goodman, Laurie; Fan, Wei; Zhang, Junqing; Li, Jun; Zhang, Juanbin; Guo, Yiran; Feng, Binxiao; Li, Heng; Lu, Yao; Fang, Xiaodong; Liang, Huiqing; Du, Zhenglin; Li, Dong; Zhao, Yiqing; Hu, Yujie; Yang, Zhenzhen; Zheng, Hancheng; Hellmann, Ines; Inouye, Michael; Pool, John; Yi, Xin; Zhao, Jing; Duan, Jinjie; Zhou, Yan; Qin, Junjie; Ma, Lijia; Li, Guoqing; Yang, Zhentao; Zhang, Guojie; Yang, Bin; Yu, Chang; Liang, Fang; Li, Wenjie; Li, Shaochuan; Li, Dawei; Ni, Peixiang; Ruan, Jue; Li, Qibin; Zhu, Hongmei; Liu, Dongyuan; Lu, Zhike; Li, Ning; Guo, Guangwu; Zhang, Jianguo; Ye, Jia; Fang, Lin; Hao, Qin; Chen, Quan; Liang, Yu; Su, Yeyang; san, A.; Ping, Cuo; Yang, Shuang; Chen, Fang; Li, Li; Zhou, Ke; Zheng, Hongkun; Ren, Yuanyuan; Yang, Ling; Gao, Yang; Yang, Guohua; Li, Zhuo; Feng, Xiaoli; Kristiansen, Karsten; Wong, Gane Ka-Shu; Nielsen, Rasmus; Durbin, Richard; Bolund, Lars; Zhang, Xiuqing; Li, Songgang; Yang, Huanming; Wang, Jian
2009-01-01
Here we present the first diploid genome sequence of an Asian individual. The genome was sequenced to 36-fold average coverage using massively parallel sequencing technology. We aligned the short reads onto the NCBI human reference genome to 99.97% coverage, and guided by the reference genome, we used uniquely mapped reads to assemble a high-quality consensus sequence for 92% of the Asian individual's genome. We identified approximately 3 million single-nucleotide polymorphisms (SNPs) inside this region, of which 13.6% were not in the dbSNP database. Genotyping analysis showed that SNP identification had high accuracy and consistency, indicating the high sequence quality of this assembly. We also carried out heterozygote phasing and haplotype prediction against HapMap CHB and JPT haplotypes (Chinese and Japanese, respectively), sequence comparison with the two available individual genomes (J. D. Watson and J. C. Venter), and structural variation identification. These variations were considered for their potential biological impact. Our sequence data and analyses demonstrate the potential usefulness of next-generation sequencing technologies for personal genomics. PMID:18987735
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Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics.
Dhouib, R; Laroche-Traineau, J; Shaha, R; Lapaillerie, D; Solier, E; Rualès, J; Pina, M; Villeneuve, P; Carrière, F; Bonneu, M; Arondel, V
2011-01-01
Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex. © 2010 The Authors Journal compilation © 2010 FEBS.
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Combining Functional and Structural Genomics to Sample the Essential Burkholderia Structome
Baugh, Loren; Gallagher, Larry A.; Patrapuvich, Rapatbhorn; Clifton, Matthew C.; Gardberg, Anna S.; Edwards, Thomas E.; Armour, Brianna; Begley, Darren W.; Dieterich, Shellie H.; Dranow, David M.; Abendroth, Jan; Fairman, James W.; Fox, David; Staker, Bart L.; Phan, Isabelle; Gillespie, Angela; Choi, Ryan; Nakazawa-Hewitt, Steve; Nguyen, Mary Trang; Napuli, Alberto; Barrett, Lynn; Buchko, Garry W.; Stacy, Robin; Myler, Peter J.; Stewart, Lance J.; Manoil, Colin; Van Voorhis, Wesley C.
2013-01-01
Background The genus Burkholderia includes pathogenic gram-negative bacteria that cause melioidosis, glanders, and pulmonary infections of patients with cancer and cystic fibrosis. Drug resistance has made development of new antimicrobials critical. Many approaches to discovering new antimicrobials, such as structure-based drug design and whole cell phenotypic screens followed by lead refinement, require high-resolution structures of proteins essential to the parasite. Methodology/Principal Findings We experimentally identified 406 putative essential genes in B. thailandensis, a low-virulence species phylogenetically similar to B. pseudomallei, the causative agent of melioidosis, using saturation-level transposon mutagenesis and next-generation sequencing (Tn-seq). We selected 315 protein products of these genes based on structure-determination criteria, such as excluding very large and/or integral membrane proteins, and entered them into the Seattle Structural Genomics Center for Infection Disease (SSGCID) structure determination pipeline. To maximize structural coverage of these targets, we applied an “ortholog rescue” strategy for those producing insoluble or difficult to crystallize proteins, resulting in the addition of 387 orthologs (or paralogs) from seven other Burkholderia species into the SSGCID pipeline. This structural genomics approach yielded structures from 31 putative essential targets from B. thailandensis, and 25 orthologs from other Burkholderia species, yielding an overall structural coverage for 49 of the 406 essential gene families, with a total of 88 depositions into the Protein Data Bank. Of these, 25 proteins have properties of a potential antimicrobial drug target i.e., no close human homolog, part of an essential metabolic pathway, and a deep binding pocket. We describe the structures of several potential drug targets in detail. Conclusions/Significance This collection of structures, solubility and experimental essentiality data provides a resource for development of drugs against infections and diseases caused by Burkholderia. All expression clones and proteins created in this study are freely available by request. PMID:23382856
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Spiliopoulou, Athina; Colombo, Marco; Orchard, Peter; Agakov, Felix; McKeigue, Paul
2017-01-01
We address the task of genotype imputation to a dense reference panel given genotype likelihoods computed from ultralow coverage sequencing as inputs. In this setting, the data have a high-level of missingness or uncertainty, and are thus more amenable to a probabilistic representation. Most existing imputation algorithms are not well suited for this situation, as they rely on prephasing for computational efficiency, and, without definite genotype calls, the prephasing task becomes computationally expensive. We describe GeneImp, a program for genotype imputation that does not require prephasing and is computationally tractable for whole-genome imputation. GeneImp does not explicitly model recombination, instead it capitalizes on the existence of large reference panels—comprising thousands of reference haplotypes—and assumes that the reference haplotypes can adequately represent the target haplotypes over short regions unaltered. We validate GeneImp based on data from ultralow coverage sequencing (0.5×), and compare its performance to the most recent version of BEAGLE that can perform this task. We show that GeneImp achieves imputation quality very close to that of BEAGLE, using one to two orders of magnitude less time, without an increase in memory complexity. Therefore, GeneImp is the first practical choice for whole-genome imputation to a dense reference panel when prephasing cannot be applied, for instance, in datasets produced via ultralow coverage sequencing. A related future application for GeneImp is whole-genome imputation based on the off-target reads from deep whole-exome sequencing. PMID:28348060
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Evaluation of the New MALDI Matrix 4-Chloro-α-Cyanocinnamic Acid
Leszyk, John D.
2010-01-01
MALDI-TOF continues to be an important tool for many proteomic studies. Recently, a new rationally designed matrix 4-chloro-α-cyanocinnamic acid was introduced, which is reported to have superior performance as compared with the “gold standard” α-cyano-4-hydroxycinnamic acid (CHCA).1 In this study, the performance of this new matrix, using the Shimadzu Biotech Axima TOF2 (Shimadzu Biotech, Manchester, UK), was investigated. The overall sequence coverage as well as sensitivity of this matrix were compared with CHCA using standard protein tryptic digests. The performance of this matrix with labile peptides, such as phosphopeptides and 4-sulfophenyl isothiocynate-derivatized peptides, to facilitate de novo sequencing was also explored. This matrix was found to be better performing than CHCA in overall sensitivity and showed better sequence coverage at low-digest levels, partly as a result of less of a bias for arginine-containing peptides. It also showed as much as a tenfold improvement in sensitivity with labile peptides on standard stainless-steel targets. In addition, as a result of the much cooler nature of this matrix, labile peptides are readily seen intact with much less fragmentation in mass spectrometry (MS) mode. This matrix was also evaluated in the MS/MS fragmentation modes of post-source decay (PSD) and collisional-induced dissociation (CID). It was found that fragmentation occurs readily in CID, however as a result of the very cool nature of this new matrix, the PSD fragments were quite weak. This matrix promises to be an important addition to the already extensive array of MALDI matrices. PMID:20592871
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A computational method for detecting copy number variations using scale-space filtering
2013-01-01
Background As next-generation sequencing technology made rapid and cost-effective sequencing available, the importance of computational approaches in finding and analyzing copy number variations (CNVs) has been amplified. Furthermore, most genome projects need to accurately analyze sequences with fairly low-coverage read data. It is urgently needed to develop a method to detect the exact types and locations of CNVs from low coverage read data. Results Here, we propose a new CNV detection method, CNV_SS, which uses scale-space filtering. The scale-space filtering is evaluated by applying to the read coverage data the Gaussian convolution for various scales according to a given scaling parameter. Next, by differentiating twice and finding zero-crossing points, inflection points of scale-space filtered read coverage data are calculated per scale. Then, the types and the exact locations of CNVs are obtained by analyzing the finger print map, the contours of zero-crossing points for various scales. Conclusions The performance of CNV_SS showed that FNR and FPR stay in the range of 1.27% to 2.43% and 1.14% to 2.44%, respectively, even at a relatively low coverage (0.5x ≤C ≤2x). CNV_SS gave also much more effective results than the conventional methods in the evaluation of FNR, at 3.82% at least and 76.97% at most even when the coverage level of read data is low. CNV_SS source code is freely available from http://dblab.hallym.ac.kr/CNV SS/. PMID:23418726
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Error and Error Mitigation in Low-Coverage Genome Assemblies
Hubisz, Melissa J.; Lin, Michael F.; Kellis, Manolis; Siepel, Adam
2011-01-01
The recent release of twenty-two new genome sequences has dramatically increased the data available for mammalian comparative genomics, but twenty of these new sequences are currently limited to ∼2× coverage. Here we examine the extent of sequencing error in these 2× assemblies, and its potential impact in downstream analyses. By comparing 2× assemblies with high-quality sequences from the ENCODE regions, we estimate the rate of sequencing error to be 1–4 errors per kilobase. While this error rate is fairly modest, sequencing error can still have surprising effects. For example, an apparent lineage-specific insertion in a coding region is more likely to reflect sequencing error than a true biological event, and the length distribution of coding indels is strongly distorted by error. We find that most errors are contributed by a small fraction of bases with low quality scores, in particular, by the ends of reads in regions of single-read coverage in the assembly. We explore several approaches for automatic sequencing error mitigation (SEM), making use of the localized nature of sequencing error, the fact that it is well predicted by quality scores, and information about errors that comes from comparisons across species. Our automatic methods for error mitigation cannot replace the need for additional sequencing, but they do allow substantial fractions of errors to be masked or eliminated at the cost of modest amounts of over-correction, and they can reduce the impact of error in downstream phylogenomic analyses. Our error-mitigated alignments are available for download. PMID:21340033
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A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies.
Utturkar, Sagar M; Klingeman, Dawn M; Hurt, Richard A; Brown, Steven D
2017-01-01
This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.
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Eaton, Deren A R; Spriggs, Elizabeth L; Park, Brian; Donoghue, Michael J
2017-05-01
Restriction-site associated DNA (RAD) sequencing and related methods rely on the conservation of enzyme recognition sites to isolate homologous DNA fragments for sequencing, with the consequence that mutations disrupting these sites lead to missing information. There is thus a clear expectation for how missing data should be distributed, with fewer loci recovered between more distantly related samples. This observation has led to a related expectation: that RAD-seq data are insufficiently informative for resolving deeper scale phylogenetic relationships. Here we investigate the relationship between missing information among samples at the tips of a tree and information at edges within it. We re-analyze and review the distribution of missing data across ten RAD-seq data sets and carry out simulations to determine expected patterns of missing information. We also present new empirical results for the angiosperm clade Viburnum (Adoxaceae, with a crown age >50 Ma) for which we examine phylogenetic information at different depths in the tree and with varied sequencing effort. The total number of loci, the proportion that are shared, and phylogenetic informativeness varied dramatically across the examined RAD-seq data sets. Insufficient or uneven sequencing coverage accounted for similar proportions of missing data as dropout from mutation-disruption. Simulations reveal that mutation-disruption, which results in phylogenetically distributed missing data, can be distinguished from the more stochastic patterns of missing data caused by low sequencing coverage. In Viburnum, doubling sequencing coverage nearly doubled the number of parsimony informative sites, and increased by >10X the number of loci with data shared across >40 taxa. Our analysis leads to a set of practical recommendations for maximizing phylogenetic information in RAD-seq studies. [hierarchical redundancy; phylogenetic informativeness; quartet informativeness; Restriction-site associated DNA (RAD) sequencing; sequencing coverage; Viburnum.]. © The authors 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.
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Complete genome sequence of the phenanthrene-degrading soil bacterium Delftia acidovorans Cs1-4
DOE Office of Scientific and Technical Information (OSTI.GOV)
Shetty, Ameesha R.; de Gannes, Vidya; Obi, Chioma C.
Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and microbial biodegradation is an important means of remediation of PAH-contaminated soil. Delftia acidovorans Cs1-4 (formerly Delftia sp. Cs1-4) was isolated by using phenanthrene as the sole carbon source from PAH contaminated soil in Wisconsin. Its full genome sequence was determined to gain insights into a mechanisms underlying biodegradation of PAH. Three genomic libraries were constructed and sequenced: an Illumina GAii shotgun library (916,416,493 reads), a 454 Titanium standard library (770,171 reads) and one paired-end 454 library (average insert size of 8 kb, 508,092 reads). The initial assembly contained 40 contigs inmore » two scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together and the consensus sequences were computationally shredded into 2 kb overlapping shreds. Illumina sequencing data was assembled, and the consensus sequence was computationally shredded into 1.5 kb overlapping shreds. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks. A total of 182 additional reactions were needed to close gaps and to raise the quality of the finished sequence. The final assembly is based on 253.3 Mb of 454 draft data (averaging 38.4 X coverage) and 590.2 Mb of Illumina draft data (averaging 89.4 X coverage). The genome of strain Cs1-4 consists of a single circular chromosome of 6,685,842 bp (66.7 %G+C) containing 6,028 predicted genes; 5,931 of these genes were protein-encoding and 4,425 gene products were assigned to a putative function. Genes encoding phenanthrene degradation were localized to a 232 kb genomic island (termed the phn island), which contained near its 3’ end a bacteriophage P4-like integrase, an enzyme often associated with chromosomal integration of mobile genetic elements. Other biodegradation pathways reconstructed from the genome sequence included: benzoate (by the acetyl-CoA pathway), styrene, nicotinic acid (by the maleamate pathway) and the pesticides Dicamba and Fenitrothion. Lastly, determination of the complete genome sequence of D. acidovorans Cs1-4 has provided new insights the microbial mechanisms of PAH biodegradation that may shape the process in the environment.« less
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Complete genome sequence of the phenanthrene-degrading soil bacterium Delftia acidovorans Cs1-4
Shetty, Ameesha R.; de Gannes, Vidya; Obi, Chioma C.; ...
2015-08-15
Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants and microbial biodegradation is an important means of remediation of PAH-contaminated soil. Delftia acidovorans Cs1-4 (formerly Delftia sp. Cs1-4) was isolated by using phenanthrene as the sole carbon source from PAH contaminated soil in Wisconsin. Its full genome sequence was determined to gain insights into a mechanisms underlying biodegradation of PAH. Three genomic libraries were constructed and sequenced: an Illumina GAii shotgun library (916,416,493 reads), a 454 Titanium standard library (770,171 reads) and one paired-end 454 library (average insert size of 8 kb, 508,092 reads). The initial assembly contained 40 contigs inmore » two scaffolds. The 454 Titanium standard data and the 454 paired end data were assembled together and the consensus sequences were computationally shredded into 2 kb overlapping shreds. Illumina sequencing data was assembled, and the consensus sequence was computationally shredded into 1.5 kb overlapping shreds. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks. A total of 182 additional reactions were needed to close gaps and to raise the quality of the finished sequence. The final assembly is based on 253.3 Mb of 454 draft data (averaging 38.4 X coverage) and 590.2 Mb of Illumina draft data (averaging 89.4 X coverage). The genome of strain Cs1-4 consists of a single circular chromosome of 6,685,842 bp (66.7 %G+C) containing 6,028 predicted genes; 5,931 of these genes were protein-encoding and 4,425 gene products were assigned to a putative function. Genes encoding phenanthrene degradation were localized to a 232 kb genomic island (termed the phn island), which contained near its 3’ end a bacteriophage P4-like integrase, an enzyme often associated with chromosomal integration of mobile genetic elements. Other biodegradation pathways reconstructed from the genome sequence included: benzoate (by the acetyl-CoA pathway), styrene, nicotinic acid (by the maleamate pathway) and the pesticides Dicamba and Fenitrothion. Lastly, determination of the complete genome sequence of D. acidovorans Cs1-4 has provided new insights the microbial mechanisms of PAH biodegradation that may shape the process in the environment.« less
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Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis.
Li, Zi-Yi; Huang, Min; Wang, Xiu-Kun; Zhu, Ying; Li, Jin-Song; Wong, Catherine C L; Fang, Qun
2018-04-17
Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.
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Highly multiplexed targeted DNA sequencing from single nuclei.
Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E
2016-02-01
Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.
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Trosman, Julia R; Weldon, Christine B; Kelley, R Kate; Phillips, Kathryn A
2015-03-01
Next-generation tumor sequencing (NGTS) panels, which include multiple established and novel targets across cancers, are emerging in oncology practice, but lack formal positive coverage by US payers. Lack of coverage may impact access and adoption. This study identified challenges of NGTS coverage by private payers. We conducted semi-structured interviews with 14 NGTS experts on potential NGTS benefits, and with 10 major payers, representing more than 125,000,000 enrollees, on NGTS coverage considerations. We used the framework approach of qualitative research for study design and thematic analyses and simple frequencies to further describe findings. All interviewed payers see potential NGTS benefits, but all noted challenges to formal coverage: 80% state that inherent features of NGTS do not fit the medical necessity definition required for coverage, 70% view NGTS as a bundle of targets versus comprehensive tumor characterization and may evaluate each target individually, and 70% express skepticism regarding new evidence methods proposed for NGTS. Fifty percent of payers expressed sufficient concerns about NGTS adoption and implementation that will preclude their ability to issue positive coverage policies. Payers perceive that NGTS holds significant promise but, in its current form, poses disruptive challenges to coverage policy frameworks. Proactive multidisciplinary efforts to define the direction for NGTS development, evidence generation, and incorporation into coverage policy are necessary to realize its promise and provide patient access. This study contributes to current literature, as possibly the first study to directly interview US payers on NGTS coverage and reimbursement. Copyright © 2015 by the National Comprehensive Cancer Network.
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Trosman, Julia R.; Weldon, Christine B.; Kate Kelley, R.; Phillips, Kathryn A.
2015-01-01
Background Next-generation tumor sequencing (NGTS) panels, which include multiple established and novel targets across cancers, are emerging in oncology practice, but lack formal positive coverage by US payers. Lack of coverage may impact access and adoption. This study identified challenges of NGTS coverage by private payers. Methods We conducted semi-structured interviews with 14 NGTS experts on potential NGTS benefits, and with 10 major payers, representing more than 125,000,000 enrollees, on NGTS coverage considerations. We used the framework approach of qualitative research for study design and thematic analyses and simple frequencies to further describe findings. Results All interviewed payers see potential NGTS benefits, but all noted challenges to formal coverage: 80% state that inherent features of NGTS do not fit the medical necessity definition required for coverage, 70% view NGTS as a bundle of targets versus comprehensive tumor characterization and may evaluate each target individually, and 70% express skepticism regarding new evidence methods proposed for NGTS. Fifty percent of payers expressed sufficient concerns about NGTS adoption and implementation that will preclude their ability to issue positive coverage policies. Conclusions Payers perceive that NGTS holds significant promise but, in its current form, poses disruptive challenges to coverage policy frameworks. Proactive multidisciplinary efforts to define the direction for NGTS development, evidence generation, and incorporation into coverage policy are necessary to realize its promise and provide patient access. This study contributes to current literature, as possibly the first study to directly interview US payers on NGTS coverage and reimbursement. PMID:25736008
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Deep whole-genome sequencing of 90 Han Chinese genomes.
Lan, Tianming; Lin, Haoxiang; Zhu, Wenjuan; Laurent, Tellier Christian Asker Melchior; Yang, Mengcheng; Liu, Xin; Wang, Jun; Wang, Jian; Yang, Huanming; Xu, Xun; Guo, Xiaosen
2017-09-01
Next-generation sequencing provides a high-resolution insight into human genetic information. However, the focus of previous studies has primarily been on low-coverage data due to the high cost of sequencing. Although the 1000 Genomes Project and the Haplotype Reference Consortium have both provided powerful reference panels for imputation, low-frequency and novel variants remain difficult to discover and call with accuracy on the basis of low-coverage data. Deep sequencing provides an optimal solution for the problem of these low-frequency and novel variants. Although whole-exome sequencing is also a viable choice for exome regions, it cannot account for noncoding regions, sometimes resulting in the absence of important, causal variants. For Han Chinese populations, the majority of variants have been discovered based upon low-coverage data from the 1000 Genomes Project. However, high-coverage, whole-genome sequencing data are limited for any population, and a large amount of low-frequency, population-specific variants remain uncharacterized. We have performed whole-genome sequencing at a high depth (∼×80) of 90 unrelated individuals of Chinese ancestry, collected from the 1000 Genomes Project samples, including 45 Northern Han Chinese and 45 Southern Han Chinese samples. Eighty-three of these 90 have been sequenced by the 1000 Genomes Project. We have identified 12 568 804 single nucleotide polymorphisms, 2 074 210 short InDels, and 26 142 structural variations from these 90 samples. Compared to the Han Chinese data from the 1000 Genomes Project, we have found 7 000 629 novel variants with low frequency (defined as minor allele frequency < 5%), including 5 813 503 single nucleotide polymorphisms, 1 169 199 InDels, and 17 927 structural variants. Using deep sequencing data, we have built a greatly expanded spectrum of genetic variation for the Han Chinese genome. Compared to the 1000 Genomes Project, these Han Chinese deep sequencing data enhance the characterization of a large number of low-frequency, novel variants. This will be a valuable resource for promoting Chinese genetics research and medical development. Additionally, it will provide a valuable supplement to the 1000 Genomes Project, as well as to other human genome projects. © The Authors 2017. Published by Oxford University Press.
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Giannone, Richard J; Wurch, Louie L; Podar, Mircea; Hettich, Robert L
2015-08-04
The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. As this interaction is thought to be membrane-associated, involving a myriad of membrane-anchored proteins, proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitans proteins. Using this method, we show that proteins with increased hydrophobic character, including membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. These gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.
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Lee, Sunghoon; Lee, Byungwook; Jang, Insoo; Kim, Sangsoo; Bhak, Jong
2006-01-01
The Localizome server predicts the transmembrane (TM) helix number and TM topology of a user-supplied eukaryotic protein and presents the result as an intuitive graphic representation. It utilizes hmmpfam to detect the presence of Pfam domains and a prediction algorithm, Phobius, to predict the TM helices. The results are combined and checked against the TM topology rules stored in a protein domain database called LocaloDom. LocaloDom is a curated database that contains TM topologies and TM helix numbers of known protein domains. It was constructed from Pfam domains combined with Swiss-Prot annotations and Phobius predictions. The Localizome server corrects the combined results of the user sequence to conform to the rules stored in LocaloDom. Compared with other programs, this server showed the highest accuracy for TM topology prediction: for soluble proteins, the accuracy and coverage were 99 and 75%, respectively, while for TM protein domain regions, they were 96 and 68%, respectively. With a graphical representation of TM topology and TM helix positions with the domain units, the Localizome server is a highly accurate and comprehensive information source for subcellular localization for soluble proteins as well as membrane proteins. The Localizome server can be found at . PMID:16845118
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Protein modelling of triterpene synthase genes from mangrove plants using Phyre2 and Swiss-model
NASA Astrophysics Data System (ADS)
Basyuni, M.; Wati, R.; Sulistiyono, N.; Hayati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.
2018-03-01
Molecular cloning of five oxidosqualene cyclases (OSC) genes from Bruguiera gymnorrhiza, Kandelia candel, and Rhizophora stylosa had previously been cloned, characterized, and encoded mono and -multi triterpene synthases. The present study analyzed protein modelling of triterpene synthase genes from mangrove using Phyre2 and Swiss-model. The diversity was noted within protein modelling of triterpene synthases using Phyre2 from sequence identity (38-43%) and residue (696-703). RsM2 was distinguishable from others for template structure; it used lanosterol synthase as a template (PDB ID: w6j.1.A). By contrast, other genes used human lanosterol synthase (1w6k.1.A). The predicted bind sites were correlated with the product of triterpene synthase, the product of BgbAS was β-amyrin, while RsM1 contained a significant amount of β-amyrin. Similarly BgLUS and KcMS, both main products was lupeol, on the other hand, RsM2 with the outcome of taraxerol. Homology modelling revealed that 696 residues of BgbAS, BgLUS, RsM1, and RsM2 (91-92% of the amino acid sequence) had been modelled with 100% confidence by the single highest scoring template using Phyre2. This coverage was higher than Swiss-model (85-90%). The present study suggested that molecular cloning of triterpene genes provides useful tools for studying the protein modelling related regulation of isoprenoids biosynthesis in mangrove forests.
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Lee, Nelson; Gatton, Michelle L.; Pelecanos, Anita; Bubb, Martin; Gonzalez, Iveth; Bell, David; Cheng, Qin
2012-01-01
Rapid diagnostic tests (RDTs) represent important tools to diagnose malaria infection. To improve understanding of the variable performance of RDTs that detect the major target in Plasmodium falciparum, namely, histidine-rich protein 2 (HRP2), and to inform the design of better tests, we undertook detailed mapping of the epitopes recognized by eight HRP-specific monoclonal antibodies (MAbs). To investigate the geographic skewing of this polymorphic protein, we analyzed the distribution of these epitopes in parasites from geographically diverse areas. To identify an ideal amino acid motif for a MAb to target in HRP2 and in the related protein HRP3, we used a purpose-designed script to perform bioinformatic analysis of 448 distinct gene sequences from pfhrp2 and from 99 sequences from the closely related gene pfhrp3. The frequency and distribution of these motifs were also compared to the MAb epitopes. Heat stability testing of MAbs immobilized on nitrocellulose membranes was also performed. Results of these experiments enabled the identification of MAbs with the most desirable characteristics for inclusion in RDTs, including copy number and coverage of target epitopes, geographic skewing, heat stability, and match with the most abundant amino acid motifs identified. This study therefore informs the selection of MAbs to include in malaria RDTs as well as in the generation of improved MAbs that should improve the performance of HRP-detecting malaria RDTs. PMID:22259210
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Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys
Berry, David; Gutierrez, Tony
2017-01-01
Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira, and Thalassolituus, as well as the Alphaproteobacterial genus Thalassospira. Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys. PMID:28567035
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Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys.
Berry, David; Gutierrez, Tony
2017-01-01
Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira , and Thalassolituus , as well as the Alphaproteobacterial genus Thalassospira . Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys.
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Seki, Takakazu; So, Christopher R; Page, Tamon R; Starkebaum, David; Hayamizu, Yuhei; Sarikaya, Mehmet
2018-02-06
The nanoscale self-organization of biomolecules, such as proteins and peptides, on solid surfaces under controlled conditions is an important issue in establishing functional bio/solid soft interfaces for bioassays, biosensors, and biofuel cells. Electrostatic interaction between proteins and surfaces is one of the most essential parameters in the adsorption and self-assembly of proteins on solid surfaces. Although the adsorption of proteins has been studied with respect to the electrochemical surface potential, the self-assembly of proteins or peptides forming well-organized nanostructures templated by lattice structure of the solid surfaces has not been studied in the relation to the surface potential. In this work, we utilize graphite-binding peptides (GrBPs) selected by the phage display method to investigate the relationship between the electrochemical potential of the highly ordered pyrolytic graphite (HOPG) and peptide self-organization forming long-range-ordered structures. Under modulated electrical bias, graphite-binding peptides form various ordered structures, such as well-ordered nanowires, dendritic structures, wavy wires, amorphous (disordered) structures, and islands. A systematic investigation of the correlation between peptide sequence and self-organizational characteristics reveals that the presence of the bias-sensitive amino acid modules in the peptide sequence has a significant effect on not only surface coverage but also on the morphological features of self-assembled structures. Our results show a new method to control peptide self-assembly by means of applied electrochemical bias as well as peptide design-rules for the construction of functional soft bio/solid interfaces that could be integrated in a wide range of practical implementations.
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Combining multiple ChIP-seq peak detection systems using combinatorial fusion.
Schweikert, Christina; Brown, Stuart; Tang, Zuojian; Smith, Phillip R; Hsu, D Frank
2012-01-01
Due to the recent rapid development in ChIP-seq technologies, which uses high-throughput next-generation DNA sequencing to identify the targets of Chromatin Immunoprecipitation, there is an increasing amount of sequencing data being generated that provides us with greater opportunity to analyze genome-wide protein-DNA interactions. In particular, we are interested in evaluating and enhancing computational and statistical techniques for locating protein binding sites. Many peak detection systems have been developed; in this study, we utilize the following six: CisGenome, MACS, PeakSeq, QuEST, SISSRs, and TRLocator. We define two methods to merge and rescore the regions of two peak detection systems and analyze the performance based on average precision and coverage of transcription start sites. The results indicate that ChIP-seq peak detection can be improved by fusion using score or rank combination. Our method of combination and fusion analysis would provide a means for generic assessment of available technologies and systems and assist researchers in choosing an appropriate system (or fusion method) for analyzing ChIP-seq data. This analysis offers an alternate approach for increasing true positive rates, while decreasing false positive rates and hence improving the ChIP-seq peak identification process.
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Wang, Yi; Coleman-Derr, Devin; Chen, Guoping; Gu, Yong Q
2015-07-01
Genome wide analysis of orthologous clusters is an important component of comparative genomics studies. Identifying the overlap among orthologous clusters can enable us to elucidate the function and evolution of proteins across multiple species. Here, we report a web platform named OrthoVenn that is useful for genome wide comparisons and visualization of orthologous clusters. OrthoVenn provides coverage of vertebrates, metazoa, protists, fungi, plants and bacteria for the comparison of orthologous clusters and also supports uploading of customized protein sequences from user-defined species. An interactive Venn diagram, summary counts, and functional summaries of the disjunction and intersection of clusters shared between species are displayed as part of the OrthoVenn result. OrthoVenn also includes in-depth views of the clusters using various sequence analysis tools. Furthermore, OrthoVenn identifies orthologous clusters of single copy genes and allows for a customized search of clusters of specific genes through key words or BLAST. OrthoVenn is an efficient and user-friendly web server freely accessible at http://probes.pw.usda.gov/OrthoVenn or http://aegilops.wheat.ucdavis.edu/OrthoVenn. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Chakraborty, Asish B.; Berger, Scott J.
2005-01-01
The balance between chromatographic performance and mass spectrometric response has been evaluated using an automated series of experiments where separations are produced by the real-time automated blending of water with organic and acidic modifiers. In this work, the concentration effects of two acidic modifiers (formic acid and trifluoroacetic acid) were studied on the separation selectivity, ultraviolet, and mass spectrometry detector response, using a complex peptide mixture. Peptide retention selectivity differences were apparent between the two modifiers, and under the conditions studied, trifluoroacetic acid produced slightly narrower (more concentrated) peaks, but significantly higher electrospray mass spectrometry suppression. Trifluoroacetic acid suppression of electrospray signal and influence on peptide retention and selectivity was dominant when mixtures of the two modifiers were analyzed. Our experimental results indicate that in analyses where the analyzed components are roughly equimolar (e.g., a peptide map of a recombinant protein), the selectivity of peptide separations can be optimized by choice and concentration of acidic modifier, without compromising the ability to obtain effective sequence coverage of a protein. In some cases, these selectivity differences were explored further, and a rational basis for differentiating acidic modifier effects from the underlying peptide sequences is described. PMID:16522853
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Lun, Aaron T.L.; Smyth, Gordon K.
2016-01-01
Chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) is widely used to identify binding sites for a target protein in the genome. An important scientific application is to identify changes in protein binding between different treatment conditions, i.e. to detect differential binding. This can reveal potential mechanisms through which changes in binding may contribute to the treatment effect. The csaw package provides a framework for the de novo detection of differentially bound genomic regions. It uses a window-based strategy to summarize read counts across the genome. It exploits existing statistical software to test for significant differences in each window. Finally, it clusters windows into regions for output and controls the false discovery rate properly over all detected regions. The csaw package can handle arbitrarily complex experimental designs involving biological replicates. It can be applied to both transcription factor and histone mark datasets, and, more generally, to any type of sequencing data measuring genomic coverage. csaw performs favorably against existing methods for de novo DB analyses on both simulated and real data. csaw is implemented as a R software package and is freely available from the open-source Bioconductor project. PMID:26578583
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Xu, Xianzhong; Pulavarti, Surya V S R K; Eletsky, Alexander; Huang, Yuanpeng Janet; Acton, Thomas B; Xiao, Rong; Everett, John K; Montelione, Gaetano T; Szyperski, Thomas
2014-12-01
High-quality solution NMR structures of three homeodomains from human proteins ALX4, ZHX1 and CASP8AP2 were solved. These domains were chosen as targets of a biomedical theme project pursued by the Northeast Structural Genomics Consortium. This project focuses on increasing the structural coverage of human proteins associated with cancer.
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USDA-ARS?s Scientific Manuscript database
The current pig reference genome sequence (Sscrofa10.2) was established using Sanger sequencing and following the clone-by-clone hierarchical shotgun sequencing approach used in the public human genome project. However, as sequence coverage was low (4-6x) the resulting assembly was only of draft qua...
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Competitive Protein Adsorption on Polysaccharide and Hyaluronate Modified Surfaces
Ombelli, Michela; Costello, Lauren; Postle, Corinne; Anantharaman, Vinod; Meng, Qing Cheng; Composto, Russell J.; Eckmann, David M.
2011-01-01
We measured adsorption of bovine serum albumin (BSA) and fibrinogen (Fg) onto six distinct bare and dextran- and hyaluronate-modified silicon surfaces created using two dextran grafting densities and three hyaluronic acid (HA) sodium salts derived from human umbilical cord, rooster comb and streptococcus zooepidemicus. Film thickness and surface morphology depended on HA molecular weight and concentration. BSA coverage was enhanced on surfaces upon competitive adsorption of BSA:Fg mixtures. Dextranization differentially reduced protein adsorption onto surfaces based on oxidation state. Hyaluronization was demonstrated to provide the greatest resistance to protein coverage, equivalent to that of the most resistant dextranized surface. Resistance to protein adsorption was independent of the type of hyaluronic acid utilized. With changing bulk protein concentration from 20 to 40 µg ml−1 for each species, Fg coverage on silicon increased by 4×, whereas both BSA and Fg adsorption on dextran and HA were far less dependent of protein bulk concentration. PMID:21623481
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Accurate and exact CNV identification from targeted high-throughput sequence data.
Nord, Alex S; Lee, Ming; King, Mary-Claire; Walsh, Tom
2011-04-12
Massively parallel sequencing of barcoded DNA samples significantly increases screening efficiency for clinically important genes. Short read aligners are well suited to single nucleotide and indel detection. However, methods for CNV detection from targeted enrichment are lacking. We present a method combining coverage with map information for the identification of deletions and duplications in targeted sequence data. Sequencing data is first scanned for gains and losses using a comparison of normalized coverage data between samples. CNV calls are confirmed by testing for a signature of sequences that span the CNV breakpoint. With our method, CNVs can be identified regardless of whether breakpoints are within regions targeted for sequencing. For CNVs where at least one breakpoint is within targeted sequence, exact CNV breakpoints can be identified. In a test data set of 96 subjects sequenced across ~1 Mb genomic sequence using multiplexing technology, our method detected mutations as small as 31 bp, predicted quantitative copy count, and had a low false-positive rate. Application of this method allows for identification of gains and losses in targeted sequence data, providing comprehensive mutation screening when combined with a short read aligner.
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Thompson, Fabiano L; Bruce, Thiago; Gonzalez, Alessandra; Cardoso, Alexander; Clementino, Maysa; Costagliola, Marcela; Hozbor, Constanza; Otero, Ernesto; Piccini, Claudia; Peressutti, Silvia; Schmieder, Robert; Edwards, Robert; Smith, Mathew; Takiyama, Luis Roberto; Vieira, Ricardo; Paranhos, Rodolfo; Artigas, Luis Felipe
2011-02-01
The bacterioplankton diversity of coastal waters along a latitudinal gradient between Puerto Rico and Argentina was analyzed using a total of 134,197 high-quality sequences from the V6 hypervariable region of the small-subunit ribosomal RNA gene (16S rRNA) (mean length of 60 nt). Most of the OTUs were identified into Proteobacteria, Bacteriodetes, Cyanobacteria, and Actinobacteria, corresponding to approx. 80% of the total number of sequences. The number of OTUs corresponding to species varied between 937 and 1946 in the seven locations. Proteobacteria appeared at high frequency in the seven locations. An enrichment of Cyanobacteria was observed in Puerto Rico, whereas an enrichment of Bacteroidetes was detected in the Argentinian shelf and Uruguayan coastal lagoons. The highest number of sequences of Actinobacteria and Acidobacteria were obtained in the Amazon estuary mouth. The rarefaction curves and Good coverage estimator for species diversity suggested a significant coverage, with values ranging between 92 and 97% for Good coverage. Conserved taxa corresponded to aprox. 52% of all sequences. This study suggests that human-contaminated environments may influence bacterioplankton diversity.
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Rare Cell Detection by Single-Cell RNA Sequencing as Guided by Single-Molecule RNA FISH.
Torre, Eduardo; Dueck, Hannah; Shaffer, Sydney; Gospocic, Janko; Gupte, Rohit; Bonasio, Roberto; Kim, Junhyong; Murray, John; Raj, Arjun
2018-02-28
Although single-cell RNA sequencing can reliably detect large-scale transcriptional programs, it is unclear whether it accurately captures the behavior of individual genes, especially those that express only in rare cells. Here, we use single-molecule RNA fluorescence in situ hybridization as a gold standard to assess trade-offs in single-cell RNA-sequencing data for detecting rare cell expression variability. We quantified the gene expression distribution for 26 genes that range from ubiquitous to rarely expressed and found that the correspondence between estimates across platforms improved with both transcriptome coverage and increased number of cells analyzed. Further, by characterizing the trade-off between transcriptome coverage and number of cells analyzed, we show that when the number of genes required to answer a given biological question is small, then greater transcriptome coverage is more important than analyzing large numbers of cells. More generally, our report provides guidelines for selecting quality thresholds for single-cell RNA-sequencing experiments aimed at rare cell analyses. Copyright © 2018 Elsevier Inc. All rights reserved.
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Adel, Susan; Kakularam, Kumar Reddy; Horn, Thomas; Reddanna, Pallu; Kuhn, Hartmut; Heydeck, Dagmar
2015-01-01
Mammalian lipoxygenases (LOXs) have been implicated in cell differentiation and in the biosynthesis of pro- and anti-inflammatory lipid mediators. The initial draft sequence of the Homo neanderthalensis genome (coverage of 1.3-fold) suggested defective leukotriene signaling in this archaic human subspecies since expression of essential proteins appeared to be corrupted. Meanwhile high quality genomic sequence data became available for two extinct human subspecies (H. neanderthalensis, Homo denisovan) and completion of the human 1000 genome project provided a comprehensive database characterizing the genetic variability of the human genome. For this study we extracted the nucleotide sequences of selected eicosanoid relevant genes (ALOX5, ALOX15, ALOX12, ALOX15B, ALOX12B, ALOXE3, COX1, COX2, LTA4H, LTC4S, ALOX5AP, CYSLTR1, CYSLTR2, BLTR1, BLTR2) from the corresponding databases. Comparison of the deduced amino acid sequences in connection with site-directed mutagenesis studies and structural modeling suggested that the major enzymes and receptors of leukotriene signaling as well as the two cyclooxygenase isoforms were fully functional in these two extinct human subspecies. Copyright © 2014 Elsevier Inc. All rights reserved.
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Asamizu, E; Nakamura, Y; Sato, S; Tabata, S
2000-06-30
For comprehensive analysis of genes expressed in the model dicotyledonous plant, Arabidopsis thaliana, expressed sequence tags (ESTs) were accumulated. Normalized and size-selected cDNA libraries were constructed from aboveground organs, flower buds, roots, green siliques and liquid-cultured seedlings, respectively, and a total of 14,026 5'-end ESTs and 39,207 3'-end ESTs were obtained. The 3'-end ESTs could be clustered into 12,028 non-redundant groups. Similarity search of the non-redundant ESTs against the public non-redundant protein database indicated that 4816 groups show similarity to genes of known function, 1864 to hypothetical genes, and the remaining 5348 are novel sequences. Gene coverage by the non-redundant ESTs was analyzed using the annotated genomic sequences of approximately 10 Mb on chromosomes 3 and 5. A total of 923 regions were hit by at least one EST, among which only 499 regions were hit by the ESTs deposited in the public database. The result indicates that the EST source generated in this project complements the EST data in the public database and facilitates new gene discovery.
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Mapping HLA-A2, -A3 and -B7 supertype-restricted T-cell epitopes in the ebolavirus proteome.
Lim, Wan Ching; Khan, Asif M
2018-01-19
Ebolavirus (EBOV) is responsible for one of the most fatal diseases encountered by mankind. Cellular T-cell responses have been implicated to be important in providing protection against the virus. Antigenic variation can result in viral escape from immune recognition. Mapping targets of immune responses among the sequence of viral proteins is, thus, an important first step towards understanding the immune responses to viral variants and can aid in the identification of vaccine targets. Herein, we performed a large-scale, proteome-wide mapping and diversity analyses of putative HLA supertype-restricted T-cell epitopes of Zaire ebolavirus (ZEBOV), the most pathogenic species among the EBOV family. All publicly available ZEBOV sequences (14,098) for each of the nine viral proteins were retrieved, removed of irrelevant and duplicate sequences, and aligned. The overall proteome diversity of the non-redundant sequences was studied by use of Shannon's entropy. The sequences were predicted, by use of the NetCTLpan server, for HLA-A2, -A3, and -B7 supertype-restricted epitopes, which are relevant to African and other ethnicities and provide for large (~86%) population coverage. The predicted epitopes were mapped to the alignment of each protein for analyses of antigenic sequence diversity and relevance to structure and function. The putative epitopes were validated by comparison with experimentally confirmed epitopes. ZEBOV proteome was generally conserved, with an average entropy of 0.16. The 185 HLA supertype-restricted T-cell epitopes predicted (82 (A2), 37 (A3) and 66 (B7)) mapped to 125 alignment positions and covered ~24% of the proteome length. Many of the epitopes showed a propensity to co-localize at select positions of the alignment. Thirty (30) of the mapped positions were completely conserved and may be attractive for vaccine design. The remaining (95) positions had one or more epitopes, with or without non-epitope variants. A significant number (24) of the putative epitopes matched reported experimentally validated HLA ligands/T-cell epitopes of A2, A3 and/or B7 supertype representative allele restrictions. The epitopes generally corresponded to functional motifs/domains and there was no correlation to localization on the protein 3D structure. These data and the epitope map provide important insights into the interaction between EBOV and the host immune system.
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Optimization of conditions to sequence long cDNAs from viruses
USDA-ARS?s Scientific Manuscript database
Fourth generation sequencing with the Minion nanopore sequencer provides opportunity to obtain deep coverage and long read for single molecules. This will benefit studies on RNA viruses. In the past, Sanger, Illumina, and Ion Torrent sequencing have been utilized to study RNA viruses. Both technique...
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Fan, Chun-Xiang; Liu, Qing-Lian; Hao, Li-Xin; Liu, Yu; Zheng, Jing-Shan; Qin, Zhi-Ying; Xia, Wei; Zhang, Shi-Yue; Yin, Zun-Dong; Jing, Qiong; Zhang, Yan-Xia; Huang, Rong-Na; Yang, Ru-Pei; Tong, Wen-Bin; Qi, Qi; Guan, Xu-Jing; Jing, Yu-Lin; Ma, Qian-Li; Wang, Jin; Ma, Xiao-Zhen; Chen, Na; Zheng, Hong-Ru; Li, Yin-Qiao; Ma, Chao; Su, Qi-Ru; Reilly, Kathleen H.; Luo, Hui-Ming; Wu, Xian-Ping; Wen, Ning; Yang, Wei-Zhong
2014-01-01
Background During August 2011–February 2012, an outbreak of type Π circulating vaccine-derived poliovirus (cVDPVs) occurred in Sichuan Province, China. Methods A field investigation of the outbreak was conducted to characterize outbreak isolates and to guide emergency response. Sequence analysis of poliovirus capsid protein VP1 was performed to determine the viral propagation, and a coverage survey was carried out for risk assessment. Results One clinical compatible polio case and three VDPV cases were determined in Ngawa County, Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province. Case patients were unimmunized children, 0.8–1 years old. Genetic sequencing showed that the isolates diverged from the VP1 region of the type Π Sabin strain by 5–12 nucleotides (nt) and shared the same 5 nt VP1 substitutions, which indicate single lineage of cVDPVs. Of the 7 acute flaccid paralysis cases (all>6 months) reported in Ngawa Prefecture in 2011, 4 (57.1%) cases (including 2 polio cases) did not receive oral attenuated poliovirus vaccine. Supplementary immunization activities (SIAs) were conducted in February–May, 2012, and the strain has not been isolated since. Conclusion High coverage of routine immunization should be maintained among children until WPV transmission is globally eradicated. Risk assessments should be conducted regularly to pinpoint high risk areas or subpopulations, with SIAs developed if necessary. PMID:25503964
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Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes.
Haiminen, Niina; Feltus, F Alex; Parida, Laxmi
2011-04-15
We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies.
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Giannone, Richard J.; Wurch, Louie L.; Podar, Mircea; ...
2015-06-25
The marine archaeon Nanoarchaeum equitans is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. It is thought that this interaction is membrane-associated, involving a myriad of membrane-anchored proteins; proteomic efforts to better characterize this difficult to analyze interface are paramount to uncovering the mechanism of their association. By extending multienzyme digestion strategies that use sample filtration to recover underdigested proteins for reprocessing/consecutive proteolytic digestion, we applied chymotrypsin to redigest the proteinaceous material left over after initial proteolysis with trypsin of sodium dodecyl sulfate (SDS)-extracted I. hospitalis-N. equitansproteins. We show that proteins with increased hydrophobic character, includingmore » membrane proteins with multiple transmembrane helices, are enriched and recovered in the underdigested fraction. Chymotryptic reprocessing provided significant sequence coverage gains in both soluble and hydrophobic proteins alike, with the latter benefiting more so in terms of membrane protein representation. Moreover, these gains were despite a large proportion of high-quality peptide spectra remaining unassigned in the underdigested fraction suggesting high levels of protein modification on these often surface-exposed proteins. Importantly, these gains were achieved without applying extensive fractionation strategies usually required for thorough characterization of membrane-associated proteins and were facilitated by the generation of a distinct, complementary set of peptides that aid in both the identification and quantitation of this important, under-represented class of proteins.« less
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Georges, Arthur; Li, Qiye; Lian, Jinmin; O'Meally, Denis; Deakin, Janine; Wang, Zongji; Zhang, Pei; Fujita, Matthew; Patel, Hardip R; Holleley, Clare E; Zhou, Yang; Zhang, Xiuwen; Matsubara, Kazumi; Waters, Paul; Graves, Jennifer A Marshall; Sarre, Stephen D; Zhang, Guojie
2015-01-01
The lizards of the family Agamidae are one of the most prominent elements of the Australian reptile fauna. Here, we present a genomic resource built on the basis of a wild-caught male ZZ central bearded dragon Pogona vitticeps. The genomic sequence for P. vitticeps, generated on the Illumina HiSeq 2000 platform, comprised 317 Gbp (179X raw read depth) from 13 insert libraries ranging from 250 bp to 40 kbp. After filtering for low-quality and duplicated reads, 146 Gbp of data (83X) was available for assembly. Exceptionally high levels of heterozygosity (0.85 % of single nucleotide polymorphisms plus sequence insertions or deletions) complicated assembly; nevertheless, 96.4 % of reads mapped back to the assembled scaffolds, indicating that the assembly included most of the sequenced genome. Length of the assembly was 1.8 Gbp in 545,310 scaffolds (69,852 longer than 300 bp), the longest being 14.68 Mbp. N50 was 2.29 Mbp. Genes were annotated on the basis of de novo prediction, similarity to the green anole Anolis carolinensis, Gallus gallus and Homo sapiens proteins, and P. vitticeps transcriptome sequence assemblies, to yield 19,406 protein-coding genes in the assembly, 63 % of which had intact open reading frames. Our assembly captured 99 % (246 of 248) of core CEGMA genes, with 93 % (231) being complete. The quality of the P. vitticeps assembly is comparable or superior to that of other published squamate genomes, and the annotated P. vitticeps genome can be accessed through a genome browser available at https://genomics.canberra.edu.au.
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Zeng, Victor; Ewen-Campen, Ben; Horch, Hadley W.; Roth, Siegfried; Mito, Taro; Extavour, Cassandra G.
2013-01-01
Most genomic resources available for insects represent the Holometabola, which are insects that undergo complete metamorphosis like beetles and flies. In contrast, the Hemimetabola (direct developing insects), representing the basal branches of the insect tree, have very few genomic resources. We have therefore created a large and publicly available transcriptome for the hemimetabolous insect Gryllus bimaculatus (cricket), a well-developed laboratory model organism whose potential for functional genetic experiments is currently limited by the absence of genomic resources. cDNA was prepared using mRNA obtained from adult ovaries containing all stages of oogenesis, and from embryo samples on each day of embryogenesis. Using 454 Titanium pyrosequencing, we sequenced over four million raw reads, and assembled them into 21,512 isotigs (predicted transcripts) and 120,805 singletons with an average coverage per base pair of 51.3. We annotated the transcriptome manually for over 400 conserved genes involved in embryonic patterning, gametogenesis, and signaling pathways. BLAST comparison of the transcriptome against the NCBI non-redundant protein database (nr) identified significant similarity to nr sequences for 55.5% of transcriptome sequences, and suggested that the transcriptome may contain 19,874 unique transcripts. For predicted transcripts without significant similarity to known sequences, we assessed their similarity to other orthopteran sequences, and determined that these transcripts contain recognizable protein domains, largely of unknown function. We created a searchable, web-based database to allow public access to all raw, assembled and annotated data. This database is to our knowledge the largest de novo assembled and annotated transcriptome resource available for any hemimetabolous insect. We therefore anticipate that these data will contribute significantly to more effective and higher-throughput deployment of molecular analysis tools in Gryllus. PMID:23671567
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Michael, Todd P; Bryant, Douglas; Gutierrez, Ryan; Borisjuk, Nikolai; Chu, Philomena; Zhang, Hanzhong; Xia, Jing; Zhou, Junfei; Peng, Hai; El Baidouri, Moaine; Ten Hallers, Boudewijn; Hastie, Alex R; Liang, Tiffany; Acosta, Kenneth; Gilbert, Sarah; McEntee, Connor; Jackson, Scott A; Mockler, Todd C; Zhang, Weixiong; Lam, Eric
2017-02-01
Spirodela polyrhiza is a fast-growing aquatic monocot with highly reduced morphology, genome size and number of protein-coding genes. Considering these biological features of Spirodela and its basal position in the monocot lineage, understanding its genome architecture could shed light on plant adaptation and genome evolution. Like many draft genomes, however, the 158-Mb Spirodela genome sequence has not been resolved to chromosomes, and important genome characteristics have not been defined. Here we deployed rapid genome-wide physical maps combined with high-coverage short-read sequencing to resolve the 20 chromosomes of Spirodela and to empirically delineate its genome features. Our data revealed a dramatic reduction in the number of the rDNA repeat units in Spirodela to fewer than 100, which is even fewer than that reported for yeast. Consistent with its unique phylogenetic position, small RNA sequencing revealed 29 Spirodela-specific microRNA, with only two being shared with Elaeis guineensis (oil palm) and Musa balbisiana (banana). Combining DNA methylation data and small RNA sequencing enabled the accurate prediction of 20.5% long terminal repeats (LTRs) that doubled the previous estimate, and revealed a high Solo:Intact LTR ratio of 8.2. Interestingly, we found that Spirodela has the lowest global DNA methylation levels (9%) of any plant species tested. Taken together our results reveal a genome that has undergone reduction, likely through eliminating non-essential protein coding genes, rDNA and LTRs. In addition to delineating the genome features of this unique plant, the methodologies described and large-scale genome resources from this work will enable future evolutionary and functional studies of this basal monocot family. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.
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Raymond, Frédéric; Boisvert, Sébastien; Roy, Gaétan; Ritt, Jean-François; Légaré, Danielle; Isnard, Amandine; Stanke, Mario; Olivier, Martin; Tremblay, Michel J.; Papadopoulou, Barbara; Ouellette, Marc; Corbeil, Jacques
2012-01-01
The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage. PMID:21998295
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An Exploration into Fern Genome Space.
Wolf, Paul G; Sessa, Emily B; Marchant, Daniel Blaine; Li, Fay-Wei; Rothfels, Carl J; Sigel, Erin M; Gitzendanner, Matthew A; Visger, Clayton J; Banks, Jo Ann; Soltis, Douglas E; Soltis, Pamela S; Pryer, Kathleen M; Der, Joshua P
2015-08-26
Ferns are one of the few remaining major clades of land plants for which a complete genome sequence is lacking. Knowledge of genome space in ferns will enable broad-scale comparative analyses of land plant genes and genomes, provide insights into genome evolution across green plants, and shed light on genetic and genomic features that characterize ferns, such as their high chromosome numbers and large genome sizes. As part of an initial exploration into fern genome space, we used a whole genome shotgun sequencing approach to obtain low-density coverage (∼0.4X to 2X) for six fern species from the Polypodiales (Ceratopteris, Pteridium, Polypodium, Cystopteris), Cyatheales (Plagiogyria), and Gleicheniales (Dipteris). We explore these data to characterize the proportion of the nuclear genome represented by repetitive sequences (including DNA transposons, retrotransposons, ribosomal DNA, and simple repeats) and protein-coding genes, and to extract chloroplast and mitochondrial genome sequences. Such initial sweeps of fern genomes can provide information useful for selecting a promising candidate fern species for whole genome sequencing. We also describe variation of genomic traits across our sample and highlight some differences and similarities in repeat structure between ferns and seed plants. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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NASA Astrophysics Data System (ADS)
He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.
2017-05-01
With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.
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NASA Astrophysics Data System (ADS)
Ismail, Ahmed E.; Grest, Gary S.; Stevens, Mark J.
2007-03-01
Oligo(ethylene oxide) self-assembled monolayers (OEO SAM's) deposited on Au are the prototypical materials used to study protein resistance. Recently, protein resistance has been shown to vary as a function of surface coverage and to be maximal at about two-thirds coverage, not complete coverage. We use molecular dynamics simulations to study the nature of the interface between water and the OEO SAM for a range of SAM coverages. As SAM coverage decreases, the amount of water within the OEO monolayer increases monotonically; however, the penetration depth of the water shows a maximum near the experimentally-found maximal coverage. As the water content increases, the SAM-water mixture becomes harder to distinguish from bulk water. Since the oxygen atoms of OEO are hydrogen bond acceptors, a hydrogen bond network forms within the SAM-water mixture. The water molecules diffuse freely within the monolayer and exchange with the bulk water. Because the monolayer becomes increasingly like bulk water as the coverage decreases, proteins stay in their bulk soluble conformation and do not adsorb. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract No. DE-AC04-94AL85000.
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Maintaining and Enhancing Diversity of Sampled Protein Conformations in Robotics-Inspired Methods.
Abella, Jayvee R; Moll, Mark; Kavraki, Lydia E
2018-01-01
The ability to efficiently sample structurally diverse protein conformations allows one to gain a high-level view of a protein's energy landscape. Algorithms from robot motion planning have been used for conformational sampling, and several of these algorithms promote diversity by keeping track of "coverage" in conformational space based on the local sampling density. However, large proteins present special challenges. In particular, larger systems require running many concurrent instances of these algorithms, but these algorithms can quickly become memory intensive because they typically keep previously sampled conformations in memory to maintain coverage estimates. In addition, robotics-inspired algorithms depend on defining useful perturbation strategies for exploring the conformational space, which is a difficult task for large proteins because such systems are typically more constrained and exhibit complex motions. In this article, we introduce two methodologies for maintaining and enhancing diversity in robotics-inspired conformational sampling. The first method addresses algorithms based on coverage estimates and leverages the use of a low-dimensional projection to define a global coverage grid that maintains coverage across concurrent runs of sampling. The second method is an automatic definition of a perturbation strategy through readily available flexibility information derived from B-factors, secondary structure, and rigidity analysis. Our results show a significant increase in the diversity of the conformations sampled for proteins consisting of up to 500 residues when applied to a specific robotics-inspired algorithm for conformational sampling. The methodologies presented in this article may be vital components for the scalability of robotics-inspired approaches.
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Pollen, Alex A; Nowakowski, Tomasz J; Shuga, Joe; Wang, Xiaohui; Leyrat, Anne A; Lui, Jan H; Li, Nianzhen; Szpankowski, Lukasz; Fowler, Brian; Chen, Peilin; Ramalingam, Naveen; Sun, Gang; Thu, Myo; Norris, Michael; Lebofsky, Ronald; Toppani, Dominique; Kemp, Darnell W; Wong, Michael; Clerkson, Barry; Jones, Brittnee N; Wu, Shiquan; Knutsson, Lawrence; Alvarado, Beatriz; Wang, Jing; Weaver, Lesley S; May, Andrew P; Jones, Robert C; Unger, Marc A; Kriegstein, Arnold R; West, Jay A A
2014-10-01
Large-scale surveys of single-cell gene expression have the potential to reveal rare cell populations and lineage relationships but require efficient methods for cell capture and mRNA sequencing. Although cellular barcoding strategies allow parallel sequencing of single cells at ultra-low depths, the limitations of shallow sequencing have not been investigated directly. By capturing 301 single cells from 11 populations using microfluidics and analyzing single-cell transcriptomes across downsampled sequencing depths, we demonstrate that shallow single-cell mRNA sequencing (~50,000 reads per cell) is sufficient for unbiased cell-type classification and biomarker identification. In the developing cortex, we identify diverse cell types, including multiple progenitor and neuronal subtypes, and we identify EGR1 and FOS as previously unreported candidate targets of Notch signaling in human but not mouse radial glia. Our strategy establishes an efficient method for unbiased analysis and comparison of cell populations from heterogeneous tissue by microfluidic single-cell capture and low-coverage sequencing of many cells.
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Deep whole-genome sequencing of 100 southeast Asian Malays.
Wong, Lai-Ping; Ong, Rick Twee-Hee; Poh, Wan-Ting; Liu, Xuanyao; Chen, Peng; Li, Ruoying; Lam, Kevin Koi-Yau; Pillai, Nisha Esakimuthu; Sim, Kar-Seng; Xu, Haiyan; Sim, Ngak-Leng; Teo, Shu-Mei; Foo, Jia-Nee; Tan, Linda Wei-Lin; Lim, Yenly; Koo, Seok-Hwee; Gan, Linda Seo-Hwee; Cheng, Ching-Yu; Wee, Sharon; Yap, Eric Peng-Huat; Ng, Pauline Crystal; Lim, Wei-Yen; Soong, Richie; Wenk, Markus Rene; Aung, Tin; Wong, Tien-Yin; Khor, Chiea-Chuen; Little, Peter; Chia, Kee-Seng; Teo, Yik-Ying
2013-01-10
Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
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Deep Whole-Genome Sequencing of 100 Southeast Asian Malays
Wong, Lai-Ping; Ong, Rick Twee-Hee; Poh, Wan-Ting; Liu, Xuanyao; Chen, Peng; Li, Ruoying; Lam, Kevin Koi-Yau; Pillai, Nisha Esakimuthu; Sim, Kar-Seng; Xu, Haiyan; Sim, Ngak-Leng; Teo, Shu-Mei; Foo, Jia-Nee; Tan, Linda Wei-Lin; Lim, Yenly; Koo, Seok-Hwee; Gan, Linda Seo-Hwee; Cheng, Ching-Yu; Wee, Sharon; Yap, Eric Peng-Huat; Ng, Pauline Crystal; Lim, Wei-Yen; Soong, Richie; Wenk, Markus Rene; Aung, Tin; Wong, Tien-Yin; Khor, Chiea-Chuen; Little, Peter; Chia, Kee-Seng; Teo, Yik-Ying
2013-01-01
Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies. PMID:23290073
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Modeling the SHG activities of diverse protein crystals
DOE Office of Scientific and Technical Information (OSTI.GOV)
Haupert, Levi M.; DeWalt, Emma L.; Simpson, Garth J.
2012-10-18
A symmetry-additiveab initiomodel for second-harmonic generation (SHG) activity of protein crystals was applied to assess the likely protein-crystal coverage of SHG microscopy. Calculations were performed for 250 proteins in nine point-group symmetries: a total of 2250 crystals. The model suggests that the crystal symmetry and the limit of detection of the instrument are expected to be the strongest predictors of coverage of the factors considered, which also included secondary-structural content and protein size. Much of the diversity in SHG activity is expected to arise primarily from the variability in the intrinsic protein response as well as the orientation within themore » crystal lattice. Two or more orders-of-magnitude variation in intensity are expected even within protein crystals of the same symmetry. SHG measurements of tetragonal lysozyme crystals confirmed detection, from which a protein coverage of ~84% was estimated based on the proportion of proteins calculated to produce SHG responses greater than that of tetragonal lysozyme. Good agreement was observed between the measured and calculated ratios of the SHG intensity from lysozyme in tetragonal and monoclinic lattices.« less
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Vukovic, Jadranka; Loftheim, Håvard; Winther, Bjørn; Reubsaet, J Léon E
2008-06-27
Off-line digestion of proteins using immobilized trypsin beads is studied with respect to the format of the digestion reactor, the digestion conditions, the comparison with in-solution digestion and its use in complex biological samples. The use of the filter vial as the most appropriate digestion reactor enables simple, efficient and easy-to-handle off-line digestion of the proteins on trypsin beads. It was shown that complex proteins like bovine serum albumin (BSA) need much longer time (89 min) and elevated temperature (37 degrees C) to be digested to an acceptable level compared to smaller proteins like cytochrome c (5 min, room temperature). Comparing the BSA digestion using immobilized trypsin beads with conventional in-solution digestion (overnight at 37 degrees C), it was shown that comparable results were obtained with respect to sequence coverage (>90%) and amount of missed cleavages (in both cases around 20 peptides with 1 or 2 missed cleavages were detected). However, the digestion using immobilized trypsin beads was considerable less time consuming. Good reproducibility and signal intensities were obtained for the digestion products of BSA in a complex urine sample. In addition to this, peptide products of proteins typically present in urine were identified.
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Martín-Campos, Trinidad; Mylonas, Roman; Masselot, Alexandre; Waridel, Patrice; Petricevic, Tanja; Xenarios, Ioannis; Quadroni, Manfredo
2017-08-04
Mass spectrometry (MS) has become the tool of choice for the large scale identification and quantitation of proteins and their post-translational modifications (PTMs). This development has been enabled by powerful software packages for the automated analysis of MS data. While data on PTMs of thousands of proteins can nowadays be readily obtained, fully deciphering the complexity and combinatorics of modification patterns even on a single protein often remains challenging. Moreover, functional investigation of PTMs on a protein of interest requires validation of the localization and the accurate quantitation of its changes across several conditions, tasks that often still require human evaluation. Software tools for large scale analyses are highly efficient but are rarely conceived for interactive, in-depth exploration of data on individual proteins. We here describe MsViz, a web-based and interactive software tool that supports manual validation of PTMs and their relative quantitation in small- and medium-size experiments. The tool displays sequence coverage information, peptide-spectrum matches, tandem MS spectra and extracted ion chromatograms through a single, highly intuitive interface. We found that MsViz greatly facilitates manual data inspection to validate PTM location and quantitate modified species across multiple samples.
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Struct2Net: a web service to predict protein–protein interactions using a structure-based approach
Singh, Rohit; Park, Daniel; Xu, Jinbo; Hosur, Raghavendra; Berger, Bonnie
2010-01-01
Struct2Net is a web server for predicting interactions between arbitrary protein pairs using a structure-based approach. Prediction of protein–protein interactions (PPIs) is a central area of interest and successful prediction would provide leads for experiments and drug design; however, the experimental coverage of the PPI interactome remains inadequate. We believe that Struct2Net is the first community-wide resource to provide structure-based PPI predictions that go beyond homology modeling. Also, most web-resources for predicting PPIs currently rely on functional genomic data (e.g. GO annotation, gene expression, cellular localization, etc.). Our structure-based approach is independent of such methods and only requires the sequence information of the proteins being queried. The web service allows multiple querying options, aimed at maximizing flexibility. For the most commonly studied organisms (fly, human and yeast), predictions have been pre-computed and can be retrieved almost instantaneously. For proteins from other species, users have the option of getting a quick-but-approximate result (using orthology over pre-computed results) or having a full-blown computation performed. The web service is freely available at http://struct2net.csail.mit.edu. PMID:20513650
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Liu, Wen-Te; Bien, Mauo-Ying; Chuang, Kai-Jen; Chang, Ta-Yuan; Jones, Tim; BéruBé, Kelly; Lalev, Georgi; Tsai, Dai-Hua; Chuang, Hsiao-Chi; Cheng, Tsun-Jen
2014-09-15
To study the toxicity of nanoparticles under relevant conditions, it is important to reproducibly disperse nanoparticles in biological media in in vitro and in vivo studies. Here, single-walled nanotubes (SWNTs) and double-walled nanotubes (DWNTs) were physicochemically and biologically characterized when dispersed in phosphate-buffered saline (PBS) and bovine serum albumin (BSA). BSA-SWNT/DWNT interaction resulted in a reduction of aggregation and an increase in particle stabilization. Based on the protein sequence coverage and protein binding results, DWNTs exhibited higher protein binding than SWNTs. SWNT and DWNT suspensions in the presence of BSA increased interleukin-6 (IL-6) levels and reduced tumor necrosis factor-alpha (TNF-α) levels in A549 cells as compared to corresponding samples in the absence of BSA. We next determined the effects of SWNTs and DWNTs on pulmonary protein modification using bronchoalveolar lavage fluid (BALF) as a surrogate collected form BALB/c mice. The BALF proteins bound to SWNTs (13 proteins) and DWNTs (11 proteins), suggesting that these proteins were associated with blood coagulation pathways. Lastly, we demonstrated the importance of physicochemical and biological alterations of SWNTs and DWNTs when dispersed in biological media, since protein binding may result in the misinterpretation of in vitro results and the activation of protein-regulated biological responses. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.
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McClory, Phillip J; Håkansson, Kristina
2017-10-03
Negative ion mode nanoelectrospray ionization (nESI) is often utilized to analyze acidic compounds, from small molecules to proteins, with mass spectrometry (MS). Under high aqueous solvent conditions, corona discharge is commonly observed at emitter tips, resulting in low ion abundances and reduced nESI needle lifetimes. We have successfully reduced corona discharge in negative ion mode by trace addition of trifluoroethanol (TFE) to aqueous samples. The addition of as little as 0.2% TFE increases aqueous spray stability not only in nESI direct infusion, but also in nanoflow liquid chromatography (nLC)/MS experiments. Negative ion mode spray stability with 0.2% TFE is approximately 6× higher than for strictly aqueous samples. Upon addition of 0.2% TFE to the mobile phase of nLC/MS experiments, tryptic peptide identifications increased from 93 to 111 peptides, resulting in an average protein sequence coverage increase of 18%.
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Lindsey, Rebecca L.; Pouseele, Hannes; Chen, Jessica C.; Strockbine, Nancy A.; Carleton, Heather A.
2016-01-01
Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS) benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org) and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x coverage, the different databases accurately predicted serotype, virulence, and resistance from WGS data, providing a fast and cheaper alternative to conventional typing techniques. PMID:27242777
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Lindsey, Rebecca L; Pouseele, Hannes; Chen, Jessica C; Strockbine, Nancy A; Carleton, Heather A
2016-01-01
Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen capable of causing severe disease in humans. Rapid and accurate identification and characterization techniques are essential during outbreak investigations. Current methods for characterization of STEC are expensive and time-consuming. With the advent of rapid and cheap whole genome sequencing (WGS) benchtop sequencers, the potential exists to replace traditional workflows with WGS. The aim of this study was to validate tools to do reference identification and characterization from WGS for STEC in a single workflow within an easy to use commercially available software platform. Publically available serotype, virulence, and antimicrobial resistance databases were downloaded from the Center for Genomic Epidemiology (CGE) (www.genomicepidemiology.org) and integrated into a genotyping plug-in with in silico PCR tools to confirm some of the virulence genes detected from WGS data. Additionally, down sampling experiments on the WGS sequence data were performed to determine a threshold for sequence coverage needed to accurately predict serotype and virulence genes using the established workflow. The serotype database was tested on a total of 228 genomes and correctly predicted from WGS for 96.1% of O serogroups and 96.5% of H serogroups identified by conventional testing techniques. A total of 59 genomes were evaluated to determine the threshold of coverage to detect the different WGS targets, 40 were evaluated for serotype and virulence gene detection and 19 for the stx gene subtypes. For serotype, 95% of the O and 100% of the H serogroups were detected at > 40x and ≥ 30x coverage, respectively. For virulence targets and stx gene subtypes, nearly all genes were detected at > 40x, though some targets were 100% detectable from genomes with coverage ≥20x. The resistance detection tool was 97% concordant with phenotypic testing results. With isolates sequenced to > 40x coverage, the different databases accurately predicted serotype, virulence, and resistance from WGS data, providing a fast and cheaper alternative to conventional typing techniques.
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EXAMINING EVIDENCE IN U.S. PAYER COVERAGE POLICIES FOR MULTI-GENE PANELS AND SEQUENCING TESTS
Chambers, James D.; Saret, Cayla J.; Anderson, Jordan E.; Deverka, Patricia A.; Douglas, Michael P.; Phillips, Kathryn A.
2017-01-01
Objectives The aim of this study was to examine the evidence payers cited in their coverage policies for multi-gene panels and sequencing tests (panels), and to compare these findings with the evidence payers cited in their coverage policies for other types of medical interventions. Methods We used the University of California at San Francisco TRANSPERS Payer Coverage Registry to identify coverage policies for panels issued by five of the largest US private payers. We reviewed each policy and categorized the evidence cited within as: clinical studies, systematic reviews, technology assessments, cost-effectiveness analyses (CEAs), budget impact studies, and clinical guidelines. We compared the evidence cited in these coverage policies for panels with the evidence cited in policies for other intervention types (pharmaceuticals, medical devices, diagnostic tests and imaging, and surgical interventions) as reported in a previous study. Results Fifty-five coverage policies for panels were included. On average, payers cited clinical guidelines in 84 percent of their coverage policies (range, 73–100 percent), clinical studies in 69 percent (50–87 percent), technology assessments 47 percent (33–86 percent), systematic reviews or meta-analyses 31 percent (7–71 percent), and CEAs 5 percent (0–7 percent). No payers cited budget impact studies in their policies. Payers less often cited clinical studies, systematic reviews, technology assessments, and CEAs in their coverage policies for panels than in their policies for other intervention types. Payers cited clinical guidelines in a comparable proportion of policies for panels and other technology types. Conclusions Payers in our sample less often cited clinical studies and other evidence types in their coverage policies for panels than they did in their coverage policies for other types of medical interventions. PMID:29065945
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EXAMINING EVIDENCE IN U.S. PAYER COVERAGE POLICIES FOR MULTI-GENE PANELS AND SEQUENCING TESTS.
Chambers, James D; Saret, Cayla J; Anderson, Jordan E; Deverka, Patricia A; Douglas, Michael P; Phillips, Kathryn A
2017-01-01
The aim of this study was to examine the evidence payers cited in their coverage policies for multi-gene panels and sequencing tests (panels), and to compare these findings with the evidence payers cited in their coverage policies for other types of medical interventions. We used the University of California at San Francisco TRANSPERS Payer Coverage Registry to identify coverage policies for panels issued by five of the largest US private payers. We reviewed each policy and categorized the evidence cited within as: clinical studies, systematic reviews, technology assessments, cost-effectiveness analyses (CEAs), budget impact studies, and clinical guidelines. We compared the evidence cited in these coverage policies for panels with the evidence cited in policies for other intervention types (pharmaceuticals, medical devices, diagnostic tests and imaging, and surgical interventions) as reported in a previous study. Fifty-five coverage policies for panels were included. On average, payers cited clinical guidelines in 84 percent of their coverage policies (range, 73-100 percent), clinical studies in 69 percent (50-87 percent), technology assessments 47 percent (33-86 percent), systematic reviews or meta-analyses 31 percent (7-71 percent), and CEAs 5 percent (0-7 percent). No payers cited budget impact studies in their policies. Payers less often cited clinical studies, systematic reviews, technology assessments, and CEAs in their coverage policies for panels than in their policies for other intervention types. Payers cited clinical guidelines in a comparable proportion of policies for panels and other technology types. Payers in our sample less often cited clinical studies and other evidence types in their coverage policies for panels than they did in their coverage policies for other types of medical interventions.
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Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer.
Court, Colin M; Ankeny, Jacob S; Sho, Shonan; Hou, Shuang; Li, Qingyu; Hsieh, Carolyn; Song, Min; Liao, Xinfang; Rochefort, Matthew M; Wainberg, Zev A; Graeber, Thomas G; Tseng, Hsian-Rong; Tomlinson, James S
2016-09-01
To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.
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Comprehensive comparison of three commercial human whole-exome capture platforms.
Asan; Xu, Yu; Jiang, Hui; Tyler-Smith, Chris; Xue, Yali; Jiang, Tao; Wang, Jiawei; Wu, Mingzhi; Liu, Xiao; Tian, Geng; Wang, Jun; Wang, Jian; Yang, Huangming; Zhang, Xiuqing
2011-09-28
Exome sequencing, which allows the global analysis of protein coding sequences in the human genome, has become an effective and affordable approach to detecting causative genetic mutations in diseases. Currently, there are several commercial human exome capture platforms; however, the relative performances of these have not been characterized sufficiently to know which is best for a particular study. We comprehensively compared three platforms: NimbleGen's Sequence Capture Array and SeqCap EZ, and Agilent's SureSelect. We assessed their performance in a variety of ways, including number of genes covered and capture efficacy. Differences that may impact on the choice of platform were that Agilent SureSelect covered approximately 1,100 more genes, while NimbleGen provided better flanking sequence capture. Although all three platforms achieved similar capture specificity of targeted regions, the NimbleGen platforms showed better uniformity of coverage and greater genotype sensitivity at 30- to 100-fold sequencing depth. All three platforms showed similar power in exome SNP calling, including medically relevant SNPs. Compared with genotyping and whole-genome sequencing data, the three platforms achieved a similar accuracy of genotype assignment and SNP detection. Importantly, all three platforms showed similar levels of reproducibility, GC bias and reference allele bias. We demonstrate key differences between the three platforms, particularly advantages of solutions over array capture and the importance of a large gene target set.
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A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies
DOE Office of Scientific and Technical Information (OSTI.GOV)
Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Jr., Richard A.
This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted.more » PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Furthermore, our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.« less
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A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies
Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Jr., Richard A.; ...
2017-07-18
This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted.more » PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Furthermore, our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.« less
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A Case Study into Microbial Genome Assembly Gap Sequences and Finishing Strategies
Utturkar, Sagar M.; Klingeman, Dawn M.; Hurt, Richard A.; Brown, Steven D.
2017-01-01
This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences. PMID:28769883
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2011-01-01
Background Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. Results A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. Conclusions The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models. PMID:21542930
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Straub, Shannon C K; Fishbein, Mark; Livshultz, Tatyana; Foster, Zachary; Parks, Matthew; Weitemier, Kevin; Cronn, Richard C; Liston, Aaron
2011-05-04
Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives. This study represents a first step in the development of a community resource for further study of plant-insect co-evolution, anti-herbivore defense, floral developmental genetics, reproductive biology, chemical evolution, population genetics, and comparative genomics using milkweeds, and A. syriaca in particular, as ecological and evolutionary models.
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Genomic Sequencing: Assessing The Health Care System, Policy, And Big-Data Implications
Phillips, Kathryn A.; Trosman, Julia; Kelley, Robin K.; Pletcher, Mark J.; Douglas, Michael P.; Weldon, Christine B.
2014-01-01
New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a “big data” technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. PMID:25006153
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Genomic sequencing: assessing the health care system, policy, and big-data implications.
Phillips, Kathryn A; Trosman, Julia R; Kelley, Robin K; Pletcher, Mark J; Douglas, Michael P; Weldon, Christine B
2014-07-01
New genomic sequencing technologies enable the high-speed analysis of multiple genes simultaneously, including all of those in a person's genome. Sequencing is a prominent example of a "big data" technology because of the massive amount of information it produces and its complexity, diversity, and timeliness. Our objective in this article is to provide a policy primer on sequencing and illustrate how it can affect health care system and policy issues. Toward this end, we developed an easily applied classification of sequencing based on inputs, methods, and outputs. We used it to examine the implications of sequencing for three health care system and policy issues: making care more patient-centered, developing coverage and reimbursement policies, and assessing economic value. We conclude that sequencing has great promise but that policy challenges include how to optimize patient engagement as well as privacy, develop coverage policies that distinguish research from clinical uses and account for bioinformatics costs, and determine the economic value of sequencing through complex economic models that take into account multiple findings and downstream costs. Project HOPE—The People-to-People Health Foundation, Inc.
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High-resolution characterization of a hepatocellular carcinoma genome.
Totoki, Yasushi; Tatsuno, Kenji; Yamamoto, Shogo; Arai, Yasuhito; Hosoda, Fumie; Ishikawa, Shumpei; Tsutsumi, Shuichi; Sonoda, Kohtaro; Totsuka, Hirohiko; Shirakihara, Takuya; Sakamoto, Hiromi; Wang, Linghua; Ojima, Hidenori; Shimada, Kazuaki; Kosuge, Tomoo; Okusaka, Takuji; Kato, Kazuto; Kusuda, Jun; Yoshida, Teruhiko; Aburatani, Hiroyuki; Shibata, Tatsuhiro
2011-05-01
Hepatocellular carcinoma, one of the most common virus-associated cancers, is the third most frequent cause of cancer-related death worldwide. By massively parallel sequencing of a primary hepatitis C virus-positive hepatocellular carcinoma (36× coverage) and matched lymphocytes (>28× coverage) from the same individual, we identified more than 11,000 somatic substitutions of the tumor genome that showed predominance of T>C/A>G transition and a decrease of the T>C substitution on the transcribed strand, suggesting preferential DNA repair. Gene annotation enrichment analysis of 63 validated non-synonymous substitutions revealed enrichment of phosphoproteins. We further validated 22 chromosomal rearrangements, generating four fusion transcripts that had altered transcriptional regulation (BCORL1-ELF4) or promoter activity. Whole-exome sequencing at a higher sequence depth (>76× coverage) revealed a TSC1 nonsense substitution in a subpopulation of the tumor cells. This first high-resolution characterization of a virus-associated cancer genome identified previously uncharacterized mutation patterns, intra-chromosomal rearrangements and fusion genes, as well as genetic heterogeneity within the tumor.
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Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes
2011-01-01
Background We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using in silico simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence. Results The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on Arabidopsis. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most. Conclusions BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies. PMID:21496274
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Licastro, Danilo; Mutarelli, Margherita; Peluso, Ivana; Neveling, Kornelia; Wieskamp, Nienke; Rispoli, Rossella; Vozzi, Diego; Athanasakis, Emmanouil; D'Eustacchio, Angela; Pizzo, Mariateresa; D'Amico, Francesca; Ziviello, Carmela; Simonelli, Francesca; Fabretto, Antonella; Scheffer, Hans; Gasparini, Paolo; Banfi, Sandro; Nigro, Vincenzo
2012-01-01
Usher syndrome (USH) is a clinically and genetically heterogeneous disorder characterized by visual and hearing impairments. Clinically, it is subdivided into three subclasses with nine genes identified so far. In the present study, we investigated whether the currently available Next Generation Sequencing (NGS) technologies are already suitable for molecular diagnostics of USH. We analyzed a total of 12 patients, most of which were negative for previously described mutations in known USH genes upon primer extension-based microarray genotyping. We enriched the NGS template either by whole exome capture or by Long-PCR of the known USH genes. The main NGS sequencing platforms were used: SOLiD for whole exome sequencing, Illumina (Genome Analyzer II) and Roche 454 (GS FLX) for the Long-PCR sequencing. Long-PCR targeting was more efficient with up to 94% of USH gene regions displaying an overall coverage higher than 25×, whereas whole exome sequencing yielded a similar coverage for only 50% of those regions. Overall this integrated analysis led to the identification of 11 novel sequence variations in USH genes (2 homozygous and 9 heterozygous) out of 18 detected. However, at least two cases were not genetically solved. Our result highlights the current limitations in the diagnostic use of NGS for USH patients. The limit for whole exome sequencing is linked to the need of a strong coverage and to the correct interpretation of sequence variations with a non obvious, pathogenic role, whereas the targeted approach suffers from the high genetic heterogeneity of USH that may be also caused by the presence of additional causative genes yet to be identified. PMID:22952768
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Recurrent hyperactive ESR1 fusion proteins in endocrine therapy-resistant breast cancer
Trabucco, S E; Priedigkeit, N; Parachoniak, C A; Vanden Borre, P; Morley, S; Rosenzweig, M; Gay, L M; Goldberg, M E; Suh, J; Ali, S M; Ross, J; Leyland-Jones, B; Young, B; Williams, C; Park, B; Tsai, M; Haley, B; Peguero, J; Callahan, R D; Sachelarie, I; Cho, J; Atkinson, J M; Bahreini, A; Nagle, A M; Puhalla, S L; Watters, R J; Erdogan-Yildirim, Z; Cao, L; Oesterreich, S; Mathew, A; Lucas, P C; Davidson, N E; Brufsky, A M; Frampton, G M; Stephens, P J; Chmielecki, J; Lee, A V
2018-01-01
Abstract Background Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287–395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3′ partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Conclusions Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6–7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies. PMID:29360925
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Fernandez-Valverde, Selene L; Calcino, Andrew D; Degnan, Bernard M
2015-05-15
The demosponge Amphimedon queenslandica is amongst the few early-branching metazoans with an assembled and annotated draft genome, making it an important species in the study of the origin and early evolution of animals. Current gene models in this species are largely based on in silico predictions and low coverage expressed sequence tag (EST) evidence. Amphimedon queenslandica protein-coding gene models are improved using deep RNA-Seq data from four developmental stages and CEL-Seq data from 82 developmental samples. Over 86% of previously predicted genes are retained in the new gene models, although 24% have additional exons; there is also a marked increase in the total number of annotated 3' and 5' untranslated regions (UTRs). Importantly, these new developmental transcriptome data reveal numerous previously unannotated protein-coding genes in the Amphimedon genome, increasing the total gene number by 25%, from 30,060 to 40,122. In general, Amphimedon genes have introns that are markedly smaller than those in other animals and most of the alternatively spliced genes in Amphimedon undergo intron-retention; exon-skipping is the least common mode of alternative splicing. Finally, in addition to canonical polyadenylation signal sequences, Amphimedon genes are enriched in a number of unique AT-rich motifs in their 3' UTRs. The inclusion of developmental transcriptome data has substantially improved the structure and composition of protein-coding gene models in Amphimedon queenslandica, providing a more accurate and comprehensive set of genes for functional and comparative studies. These improvements reveal the Amphimedon genome is comprised of a remarkably high number of tightly packed genes. These genes have small introns and there is pervasive intron retention amongst alternatively spliced transcripts. These aspects of the sponge genome are more similar unicellular opisthokont genomes than to other animal genomes.
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The Effects of Nucleosome Positioning and Chromatin Architecture on Transgene Expression
NASA Astrophysics Data System (ADS)
Kempton, Colton E.
Eukaryotes use proteins to carefully package and compact their genomes to fit into the nuclei of their individual cells. Nucleosomes are the primary level of compaction. Nucleosomes are formed when DNA wraps around an octamer of histone proteins and a nucleosome's position can limit access to genetic regulatory elements. Therefore, nucleosomes represent a basic level of gene regulation. DNA and its associated proteins, called chromatin, is usually classified as euchromatin or heterochromatin. Euchromatin is transcriptionally active with loosely packed nucleosomes while heterochromatin is condensed with tightly packed nucleosomes and is transcriptionally silent. In order to become active, heterochromatin must first be remodeled. We have studied the effects of nucleosome positioning on transgene expression in vivo using Caenorhabditis elegans as a model. We show that both location and polarity of the DNA sequence can influence transgene expression. We also discuss some considerations for working with CRISPR/Cas9. A major reason for doing in vitro nucleosome reconstitutions is to determine the effects of DNA sequence on nucleosome formation and position. It has previously been implied that nucleosome reconstitutions are stochastic and not very reproducible. We show that nucleosome reconstitutions are highly reproducible under our reaction conditions. Our results also indicate that a minimum depth of 35X sequencing coverage be maintained for maximal gains in Pearson's correlation coefficients. Communicating science with others is an important skill for any researcher. The rising generation of scientists need mentors who can teach them how to be independent thinkers who can carry out scientific experiments and communicate their finding to others. With this goal in mind, we have devised a scaffolding pedagogical method to help transform undergraduates into confident independent thinkers and researchers.
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Comprehensive Characterization of Swine Cardiac Troponin T Proteoforms by Top-Down Mass Spectrometry
NASA Astrophysics Data System (ADS)
Lin, Ziqing; Guo, Fang; Gregorich, Zachery R.; Sun, Ruixiang; Zhang, Han; Hu, Yang; Shanmuganayagam, Dhanansayan; Ge, Ying
2018-04-01
Cardiac troponin T (cTnT) regulates the Ca2+-mediated interaction between myosin thick filaments and actin thin filaments during cardiac contraction and relaxation. cTnT is released into the blood following injury, and increased serum levels of the protein are used clinically as a biomarker for myocardial infarction. Moreover, mutations in cTnT are causative in a number of familial cardiomyopathies. With the increasing use of large animal (swine) model to recapitulate human diseases, it is essential to characterize species-dependent protein sequence variants, alternative RNA splicing, and post-translational modifications (PTMs), but challenges remain due to the incomplete database and lack of validation of the predicted splicing isoforms. Herein, we integrated top-down mass spectrometry (MS) with online liquid chromatography (LC) and immunoaffinity purification to comprehensively characterize miniature swine cTnT proteoforms, including those arising from alternative RNA splicing and PTMs. A total of seven alternative splicing isoforms of cTnT were identified by LC/MS from swine left ventricular tissue, with each isoform containing un-phosphorylated and mono-phosphorylated proteoforms. The phosphorylation site was localized to Ser1 for the mono-phosphorylated proteoforms of cTnT1, 3, 4, and 6 by online MS/MS combining collisionally activated dissociation (CAD) and electron transfer dissociation (ETD). Offline MS/MS on Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometer with CAD and electron capture dissociation (ECD) was then utilized to achieve deep sequencing of mono-phosphorylated cTnT1 (35.2 kDa) with a high sequence coverage of 87%. Taken together, this study demonstrated the unique advantage of top-down MS in the comprehensive characterization of protein alternative splicing isoforms together with PTMs. [Figure not available: see fulltext.
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Rescuing discarded spectra: Full comprehensive analysis of a minimal proteome.
Lluch-Senar, Maria; Mancuso, Francesco M; Climente-González, Héctor; Peña-Paz, Marcia I; Sabido, Eduard; Serrano, Luis
2016-02-01
A common problem encountered when performing large-scale MS proteome analysis is the loss of information due to the high percentage of unassigned spectra. To determine the causes behind this loss we have analyzed the proteome of one of the smallest living bacteria that can be grown axenically, Mycoplasma pneumoniae (729 ORFs). The proteome of M. pneumoniae cells, grown in defined media, was analyzed by MS. An initial search with both Mascot and a species-specific NCBInr database with common contaminants (NCBImpn), resulted in around 79% of the acquired spectra not having an assignment. The percentage of non-assigned spectra was reduced to 27% after re-analysis of the data with the PEAKS software, thereby increasing the proteome coverage of M. pneumoniae from the initial 60% to over 76%. Nonetheless, 33,413 spectra with assigned amino acid sequences could not be mapped to any NCBInr database protein sequence. Approximately, 1% of these unassigned peptides corresponded to PTMs and 4% to M. pneumoniae protein variants (deamidation and translation inaccuracies). The most abundant peptide sequence variants (Phe-Tyr and Ala-Ser) could be explained by alterations in the editing capacity of the corresponding tRNA synthases. About another 1% of the peptides not associated to any protein had repetitions of the same aromatic/hydrophobic amino acid at the N-terminus, or had Arg/Lys at the C-terminus. Thus, in a model system, we have maximized the number of assigned spectra to 73% (51,453 out of the 70,040 initial acquired spectra). All MS data have been deposited in the ProteomeXchange with identifier PXD002779 (http://proteomecentral.proteomexchange.org/dataset/PXD002779). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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The complete mitochondrial genome of the sandbar shark Carcharhinus plumbeus.
Blower, Dean C; Ovenden, Jennifer R
2016-01-01
The sandbar shark, Carcharhinus plumbeus, a major representative species in shark fisheries worldwide is now considered vulnerable to overfishing. A pool of 774,234 Roche 454 shotgun sequences from one individual were assembled into a 16,706 bp mitogenome with 33× average coverage depth. It comprised 13 protein coding genes, 22 transfer RNA's, 2 ribosomal genes and 2 non-coding regions, typical of a vertebrate mitogenome. As expected for sharks, an A-T nucleotide bias was evident. This adds to rapidly growing number of mitogenome assemblies for the economically important Carcharhinidae family. The C. plumbeus mitogenome will assist researchers, fisheries and conservation managers interested in shark molecular systematics, phylogeography, conservation genetics, population and stock structure.
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Insights into metazoan evolution from alvinella pompejana cDNAs
2010-01-01
Background Alvinella pompejana is a representative of Annelids, a key phylum for evo-devo studies that is still poorly studied at the sequence level. A. pompejana inhabits deep-sea hydrothermal vents and is currently known as one of the most thermotolerant Eukaryotes in marine environments, withstanding the largest known chemical and thermal ranges (from 5 to 105°C). This tube-dwelling worm forms dense colonies on the surface of hydrothermal chimneys and can withstand long periods of hypo/anoxia and long phases of exposure to hydrogen sulphides. A. pompejana specifically inhabits chimney walls of hydrothermal vents on the East Pacific Rise. To survive, Alvinella has developed numerous adaptations at the physiological and molecular levels, such as an increase in the thermostability of proteins and protein complexes. It represents an outstanding model organism for studying adaptation to harsh physicochemical conditions and for isolating stable macromolecules resistant to high temperatures. Results We have constructed four full length enriched cDNA libraries to investigate the biology and evolution of this intriguing animal. Analysis of more than 75,000 high quality reads led to the identification of 15,858 transcripts and 9,221 putative protein sequences. Our annotation reveals a good coverage of most animal pathways and networks with a prevalence of transcripts involved in oxidative stress resistance, detoxification, anti-bacterial defence, and heat shock protection. Alvinella proteins seem to show a slow evolutionary rate and a higher similarity with proteins from Vertebrates compared to proteins from Arthropods or Nematodes. Their composition shows enrichment in positively charged amino acids that might contribute to their thermostability. The gene content of Alvinella reveals that an important pool of genes previously considered to be specific to Deuterostomes were in fact already present in the last common ancestor of the Bilaterian animals, but have been secondarily lost in model invertebrates. This pool is enriched in glycoproteins that play a key role in intercellular communication, hormonal regulation and immunity. Conclusions Our study starts to unravel the gene content and sequence evolution of a deep-sea annelid, revealing key features in eukaryote adaptation to extreme environmental conditions and highlighting the proximity of Annelids and Vertebrates. PMID:21080938
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Yamaguchi, Akihiro; Go, Mitiko
2006-01-01
We have been developing FAMSBASE, a protein homology-modeling database of whole ORFs predicted from genome sequences. The latest update of FAMSBASE (http://daisy.nagahama-i-bio.ac.jp/Famsbase/), which is based on the protein three-dimensional (3D) structures released by November 2003, contains modeled 3D structures for 368,724 open reading frames (ORFs) derived from genomes of 276 species, namely 17 archaebacterial, 130 eubacterial, 18 eukaryotic and 111 phage genomes. Those 276 genomes are predicted to have 734,193 ORFs in total and the current FAMSBASE contains protein 3D structure of approximately 50% of the ORF products. However, cases that a modeled 3D structure covers the whole part of an ORF product are rare. When portion of an ORF with 3D structure is compared in three kingdoms of life, in archaebacteria and eubacteria, approximately 60% of the ORFs have modeled 3D structures covering almost the entire amino acid sequences, however, the percentage falls to about 30% in eukaryotes. When annual differences in the number of ORFs with modeled 3D structure are calculated, the fraction of modeled 3D structures of soluble protein for archaebacteria is increased by 5%, and that for eubacteria by 7% in the last 3 years. Assuming that this rate would be maintained and that determination of 3D structures for predicted disordered regions is unattainable, whole soluble protein model structures of prokaryotes without the putative disordered regions will be in hand within 15 years. For eukaryotic proteins, they will be in hand within 25 years. The 3D structures we will have at those times are not the 3D structure of the entire proteins encoded in single ORFs, but the 3D structures of separate structural domains. Measuring or predicting spatial arrangements of structural domains in an ORF will then be a coming issue of structural genomics. PMID:17146617
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Evaluating imputation algorithms for low-depth genotyping-by-sequencing (GBS) data
USDA-ARS?s Scientific Manuscript database
Well-powered genomic studies require genome-wide marker coverage across many individuals. For non-model species with few genomic resources, high-throughput sequencing (HTS) methods, such as Genotyping-By-Sequencing (GBS), offer an inexpensive alternative to array-based genotyping. Although affordabl...
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Optimization of parameters for coverage of low molecular weight proteins
Müller, Stephan A.; Kohajda, Tibor; Findeiß, Sven; Stadler, Peter F.; Washietl, Stefan; Kellis, Manolis; von Bergen, Martin
2010-01-01
Proteins with molecular weights of <25 kDa are involved in major biological processes such as ribosome formation, stress adaption (e.g., temperature reduction) and cell cycle control. Despite their importance, the coverage of smaller proteins in standard proteome studies is rather sparse. Here we investigated biochemical and mass spectrometric parameters that influence coverage and validity of identification. The underrepresentation of low molecular weight (LMW) proteins may be attributed to the low numbers of proteolytic peptides formed by tryptic digestion as well as their tendency to be lost in protein separation and concentration/desalting procedures. In a systematic investigation of the LMW proteome of Escherichia coli, a total of 455 LMW proteins (27% of the 1672 listed in the SwissProt protein database) were identified, corresponding to a coverage of 62% of the known cytosolic LMW proteins. Of these proteins, 93 had not yet been functionally classified, and five had not previously been confirmed at the protein level. In this study, the influences of protein extraction (either urea or TFA), proteolytic digestion (solely, and the combined usage of trypsin and AspN as endoproteases) and protein separation (gel- or non-gel-based) were investigated. Compared to the standard procedure based solely on the use of urea lysis buffer, in-gel separation and tryptic digestion, the complementary use of TFA for extraction or endoprotease AspN for proteolysis permits the identification of an extra 72 (32%) and 51 proteins (23%), respectively. Regarding mass spectrometry analysis with an LTQ Orbitrap mass spectrometer, collision-induced fragmentation (CID and HCD) and electron transfer dissociation using the linear ion trap (IT) or the Orbitrap as the analyzer were compared. IT-CID was found to yield the best identification rate, whereas IT-ETD provided almost comparable results in terms of LMW proteome coverage. The high overlap between the proteins identified with IT-CID and IT-ETD allowed the validation of 75% of the identified proteins using this orthogonal fragmentation technique. Furthermore, a new approach to evaluating and improving the completeness of protein databases that utilizes the program RNAcode was introduced and examined. Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-4093-x) contains supplementary material, which is available to authorized users. PMID:20803007
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EEG microstates during resting represent personality differences.
Schlegel, Felix; Lehmann, Dietrich; Faber, Pascal L; Milz, Patricia; Gianotti, Lorena R R
2012-01-01
We investigated the spontaneous brain electric activity of 13 skeptics and 16 believers in paranormal phenomena; they were university students assessed with a self-report scale about paranormal beliefs. 33-channel EEG recordings during no-task resting were processed as sequences of momentary potential distribution maps. Based on the maps at peak times of Global Field Power, the sequences were parsed into segments of quasi-stable potential distribution, the 'microstates'. The microstates were clustered into four classes of map topographies (A-D). Analysis of the microstate parameters time coverage, occurrence frequency and duration as well as the temporal sequence (syntax) of the microstate classes revealed significant differences: Believers had a higher coverage and occurrence of class B, tended to decreased coverage and occurrence of class C, and showed a predominant sequence of microstate concatenations from A to C to B to A that was reversed in skeptics (A to B to C to A). Microstates of different topographies, putative "atoms of thought", are hypothesized to represent different types of information processing.The study demonstrates that personality differences can be detected in resting EEG microstate parameters and microstate syntax. Microstate analysis yielded no conclusive evidence for the hypothesized relation between paranormal belief and schizophrenia.
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A case of canine borreliosis in Iran caused by Borrelia persica.
Shirani, Darush; Rakhshanpoor, Alaleh; Cutler, Sally Jane; Ghazinezhad, Behnaz; Naddaf, Saied Reza
2016-04-01
Tick-borne relapsing fever is an endemic disease in Iran, with most cases attributed to infection by Borrelia persica, which is transmitted by Ornithodoros tholozani soft ticks. Here, we report spirochetemia in blood of a puppy residing in Tehran, Iran. The causative species was identified by use of highly discriminative IGS sequencing; the 489 bp IGS sequence obtained in our study showed 99% identity (100% coverage) when compared with B. persica sequences derived from clinical cases or from O. tholozani ticks. Our IGS sequence also showed 99% similarity over 414 bp (85% coverage) with a strain from a domestic dog, and 96% over 328 bp (69% coverage) with a strain from a domestic cat. Pet-keeping in cosmopolitan cities like Tehran has become increasingly popular in recent years. Animals are often transported into the city in cages or cardboard boxes that might also harbor minute tick larvae and/or early stages of the nymphs bringing them into the urban environment. This may pose a threat to household members who buy and keep these puppies and as a result may come into close contact with infected ticks. Copyright © 2016 Elsevier GmbH. All rights reserved.
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ReadXplorer—visualization and analysis of mapped sequences
Hilker, Rolf; Stadermann, Kai Bernd; Doppmeier, Daniel; Kalinowski, Jörn; Stoye, Jens; Straube, Jasmin; Winnebald, Jörn; Goesmann, Alexander
2014-01-01
Motivation: Fast algorithms and well-arranged visualizations are required for the comprehensive analysis of the ever-growing size of genomic and transcriptomic next-generation sequencing data. Results: ReadXplorer is a software offering straightforward visualization and extensive analysis functions for genomic and transcriptomic DNA sequences mapped on a reference. A unique specialty of ReadXplorer is the quality classification of the read mappings. It is incorporated in all analysis functions and displayed in ReadXplorer's various synchronized data viewers for (i) the reference sequence, its base coverage as (ii) normalizable plot and (iii) histogram, (iv) read alignments and (v) read pairs. ReadXplorer's analysis capability covers RNA secondary structure prediction, single nucleotide polymorphism and deletion–insertion polymorphism detection, genomic feature and general coverage analysis. Especially for RNA-Seq data, it offers differential gene expression analysis, transcription start site and operon detection as well as RPKM value and read count calculations. Furthermore, ReadXplorer can combine or superimpose coverage of different datasets. Availability and implementation: ReadXplorer is available as open-source software at http://www.readxplorer.org along with a detailed manual. Contact: rhilker@mikrobio.med.uni-giessen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24790157
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Protein cleavage strategies for an improved analysis of the membrane proteome
Fischer, Frank; Poetsch, Ansgar
2006-01-01
Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms. PMID:16512920
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Protective Immunogenicity of Group A Streptococcal M-Related Proteins
Niedermeyer, Shannon E.; Agbaosi, Tina; Hysmith, Nicholas D.; Penfound, Thomas A.; Hohn, Claudia M.; Pullen, Matthew; Bright, Michael I.; Murrell, Daniel S.; Shenep, Lori E.; Courtney, Harry S.
2015-01-01
Many previous studies have focused on the surface M proteins of group A streptococci (GAS) as virulence determinants and protective antigens. However, the majority of GAS isolates express M-related protein (Mrp) in addition to M protein, and both have been shown to be required for optimal virulence. In the current study, we evaluated the protective immunogenicity of Mrp to determine its potential as a vaccine component that may broaden the coverage of M protein-based vaccines. Sequence analyses of 33 mrp genes indicated that there are three families of structurally related Mrps (MrpI, MrpII, and MrpIII). N-terminal peptides of Mrps were cloned, expressed, and purified from M type 2 (M2) (MrpI), M4 (MrpII), and M49 (MrpIII) GAS. Rabbit antisera against the Mrps reacted at high titers with the homologous Mrp, as determined by enzyme-linked immunosorbent assay, and promoted bactericidal activity against GAS emm types expressing Mrps within the same family. Mice passively immunized with rabbit antisera against MrpII were protected against challenge infections with M28 GAS. Assays for Mrp antibodies in serum samples from 281 pediatric subjects aged 2 to 16 indicated that the Mrp immune response correlated with increasing age of the subjects. Affinity-purified human Mrp antibodies promoted bactericidal activity against a number of GAS representing different emm types that expressed an Mrp within the same family but showed no activity against emm types expressing an Mrp from a different family. Our results indicate that Mrps have semiconserved N-terminal sequences that contain bactericidal epitopes which are immunogenic in humans. These findings may have direct implications for the development of GAS vaccines. PMID:25630406
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Keel, B N; Nonneman, D J; Rohrer, G A
2017-08-01
Genetic variants detected from sequence have been used to successfully identify causal variants and map complex traits in several organisms. High and moderate impact variants, those expected to alter or disrupt the protein coded by a gene and those that regulate protein production, likely have a more significant effect on phenotypic variation than do other types of genetic variants. Hence, a comprehensive list of these functional variants would be of considerable interest in swine genomic studies, particularly those targeting fertility and production traits. Whole-genome sequence was obtained from 72 of the founders of an intensely phenotyped experimental swine herd at the U.S. Meat Animal Research Center (USMARC). These animals included all 24 of the founding boars (12 Duroc and 12 Landrace) and 48 Yorkshire-Landrace composite sows. Sequence reads were mapped to the Sscrofa10.2 genome build, resulting in a mean of 6.1 fold (×) coverage per genome. A total of 22 342 915 high confidence SNPs were identified from the sequenced genomes. These included 21 million previously reported SNPs and 79% of the 62 163 SNPs on the PorcineSNP60 BeadChip assay. Variation was detected in the coding sequence or untranslated regions (UTRs) of 87.8% of the genes in the porcine genome: loss-of-function variants were predicted in 504 genes, 10 202 genes contained nonsynonymous variants, 10 773 had variation in UTRs and 13 010 genes contained synonymous variants. Approximately 139 000 SNPs were classified as loss-of-function, nonsynonymous or regulatory, which suggests that over 99% of the variation detected in our pigs could potentially be ignored, allowing us to focus on a much smaller number of functional SNPs during future analyses. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.
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Ullah, Ihsan; Jang, Eun-Kyung; Kim, Min-Sung; Shin, Jin-Ho; Park, Gun-Seok; Khan, Abdur Rahim; Hong, Sung-Jun; Jung, Byung-Kwon; Choi, JungBae; Park, YeongJun; Kwak, Yunyoung; Shin, Jae-Ho
2014-01-01
Photorhabdus temperata is an entomopathogenic enterobacterium; it is a nematode symbiont that possesses pathogenicity islands involved in insect virulence. Herein, we constructed a P. temperata M1021 cosmid library in Escherichia coli XL1-Blue MRF` and obtained 7.14 × 105 clones. However, only 1020 physiologically active clones were screened for insect virulence factors by injection of each E. coli cosmid clone into Galleria mellonella and Tenebrio molitor larvae. A single cosmid clone, PtC1015, was consequently selected due to its characteristic virulent properties, e.g., loss of body turgor followed by death of larvae when the clone was injected into the hemocoel. The sequence alignment against the available sequences in Swiss-Prot and NCBI databases, confirmed the presence of the mcf gene homolog in the genome of P. temperata M1021 showing 85% homology and 98% query coverage with the P. luminescens counterpart. Furthermore, a 2932 amino acid long Mcf protein revealed limited similarity with three protein domains. The N-terminus of the Mcf encompassed consensus sequence for a BH3 domain, the central region revealed similarity to toxin B, and the C-terminus of Mcf revealed similarity to the bacterial export domain of ApxIVA, an RTX-like toxin. In short, the Mcf toxin is likely to play a role in the elimination of insect pests, making it a promising model for use in the agricultural field. PMID:25014195
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HetMappsS: Heterozygous mapping strategy for high resolution Genotyping-by-Sequencing Markers
USDA-ARS?s Scientific Manuscript database
Reduced representation genotyping approaches, such as genotyping-by-sequencing (GBS), provide opportunities to generate high-resolution genetic maps at a low per-sample cost. However, missing data and non-uniform sequence coverage can complicate map creation in highly heterozygous species. To facili...
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Laskin, Julia [Richland, WA; Futrell, Jean H [Richland, WA
2008-04-29
The invention relates to a method and apparatus for enhanced sequencing of complex molecules using surface-induced dissociation (SID) in conjunction with mass spectrometric analysis. Results demonstrate formation of a wide distribution of structure-specific fragments having wide sequence coverage useful for sequencing and identifying the complex molecules.
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Ciotlos, Serban; Mao, Qing; Zhang, Rebecca Yu; Li, Zhenyu; Chin, Robert; Gulbahce, Natali; Liu, Sophie Jia; Drmanac, Radoje; Peters, Brock A
2016-01-01
The cell line BT-474 is a popular cell line for studying the biology of cancer and developing novel drugs. However, there is no complete, published genome sequence for this highly utilized scientific resource. In this study we sought to provide a comprehensive and useful data set for the scientific community by generating a whole genome sequence for BT-474. Five μg of genomic DNA, isolated from an early passage of the BT-474 cell line, was used to generate a whole genome sequence (114X coverage) using Complete Genomics' standard sequencing process. To provide additional variant phasing and structural variation data we also processed and analyzed two separate libraries of 5 and 6 individual cells to depths of 99X and 87X, respectively, using Complete Genomics' Long Fragment Read (LFR) technology. BT-474 is a highly aneuploid cell line with an extremely complex genome sequence. This ~300X total coverage genome sequence provides a more complete understanding of this highly utilized cell line at the genomic level.
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Budiman, Muhammad A.; Mao, Long; Wood, Todd C.; Wing, Rod A.
2000-01-01
Recently a new strategy using BAC end sequences as sequence-tagged connectors (STCs) was proposed for whole-genome sequencing projects. In this study, we present the construction and detailed characterization of a 15.0 haploid genome equivalent BAC library for the cultivated tomato, Lycopersicon esculentum cv. Heinz 1706. The library contains 129,024 clones with an average insert size of 117.5 kb and a chloroplast content of 1.11%. BAC end sequences from 1490 ends were generated and analyzed as a preliminary evaluation for using this library to develop an STC framework to sequence the tomato genome. A total of 1205 BAC end sequences (80.9%) were obtained, with an average length of 360 high-quality bases, and were searched against the GenBank database. Using a cutoff expectation value of <10−6, and combining the results from BLASTN, BLASTX, and TBLASTX searches, 24.3% of the BAC end sequences were similar to known sequences, of which almost half (48.7%) share sequence similarities to retrotransposons and 7% to known genes. Some of the transposable element sequences were the first reported in tomato, such as sequences similar to maize transposon Activator (Ac) ORF and tobacco pararetrovirus-like sequences. Interestingly, there were no BAC end sequences similar to the highly repeated TGRI and TGRII elements. However, the majority (70.3%) of STCs did not share significant sequence similarities to any sequences in GenBank at either the DNA or predicted protein levels, indicating that a large portion of the tomato genome is still unknown. Our data demonstrate that this BAC library is suitable for developing an STC database to sequence the tomato genome. The advantages of developing an STC framework for whole-genome sequencing of tomato are discussed. [The BAC end sequences described in this paper have been deposited in the GenBank data library under accession nos. AQ367111–AQ368361.] PMID:10645957
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Kariuki, Simon K; Njunge, James; Muia, Ann; Muluvi, Geofrey; Gatei, Wangeci; Ter Kuile, Feiko; Terlouw, Dianne J; Hawley, William A; Phillips-Howard, Penelope A; Nahlen, Bernard L; Lindblade, Kim A; Hamel, Mary J; Slutsker, Laurence; Shi, Ya Ping
2013-08-27
Although several studies have investigated the impact of reduced malaria transmission due to insecticide-treated bed nets (ITNs) on the patterns of morbidity and mortality, there is limited information on their effect on parasite diversity. Sequencing was used to investigate the effect of ITNs on polymorphisms in two genes encoding leading Plasmodium falciparum vaccine candidate antigens, the 19 kilodalton blood stage merozoite surface protein-1 (MSP-1(19kDa)) and the Th2R and Th3R T-cell epitopes of the pre-erythrocytic stage circumsporozoite protein (CSP) in a large community-based ITN trial site in western Kenya. The number and frequency of haplotypes as well as nucleotide and haplotype diversity were compared among parasites obtained from children <5 years old prior to the introduction of ITNs (1996) and after 5 years of high coverage ITN use (2001). A total of 12 MSP-1(19kDa) haplotypes were detected in 1996 and 2001. The Q-KSNG-L and E-KSNG-L haplotypes corresponding to the FVO and FUP strains of P. falciparum were the most prevalent (range 32-37%), with an overall haplotype diversity of > 0.7. No MSP-1(19kDa) 3D7 sequence-types were detected in 1996 and the frequency was less than 4% in 2001. The CSP Th2R and Th3R domains were highly polymorphic with a total of 26 and 14 haplotypes, respectively detected in 1996 and 34 and 13 haplotypes in 2001, with an overall haplotype diversity of > 0.9 and 0.75 respectively. The frequency of the most predominant Th2R and Th3R haplotypes was 14 and 36%, respectively. The frequency of Th2R and Th3R haplotypes corresponding to the 3D7 parasite strain was less than 4% at both time points. There was no significant difference in nucleotide and haplotype diversity in parasite isolates collected at both time points. High diversity in these two genes has been maintained overtime despite marked reductions in malaria transmission due to ITNs use. The frequency of 3D7 sequence-types was very low in this area. These findings provide information that could be useful in the design of future malaria vaccines for deployment in endemic areas with high ITN coverage and in interpretation of efficacy data for malaria vaccines based on 3D7 parasite strains.
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Marine, Rachel; McCarren, Coleen; Vorrasane, Vansay; Nasko, Dan; Crowgey, Erin; Polson, Shawn W; Wommack, K Eric
2014-01-30
Shotgun metagenomics has become an important tool for investigating the ecology of microorganisms. Underlying these investigations is the assumption that metagenome sequence data accurately estimates the census of microbial populations. Multiple displacement amplification (MDA) of microbial community DNA is often used in cases where it is difficult to obtain enough DNA for sequencing; however, MDA can result in amplification biases that may impact subsequent estimates of population census from metagenome data. Some have posited that pooling replicate MDA reactions negates these biases and restores the accuracy of population analyses. This assumption has not been empirically tested. Using mock viral communities, we examined the influence of pooling on population-scale analyses. In pooled and single reaction MDA treatments, sequence coverage of viral populations was highly variable and coverage patterns across viral genomes were nearly identical, indicating that initial priming biases were reproducible and that pooling did not alleviate biases. In contrast, control unamplified sequence libraries showed relatively even coverage across phage genomes. MDA should be avoided for metagenomic investigations that require quantitative estimates of microbial taxa and gene functional groups. While MDA is an indispensable technique in applications such as single-cell genomics, amplification biases cannot be overcome by combining replicate MDA reactions. Alternative library preparation techniques should be utilized for quantitative microbial ecology studies utilizing metagenomic sequencing approaches.
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Sequenced sorghum mutant library- an efficient platform for discovery of causal gene mutations
USDA-ARS?s Scientific Manuscript database
Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. We applied whole-genome sequencing to 256 phenotyped mutant lines of sorghum (Sorghum bicolor L. Moench) to 16x coverage. Comparisons with the reference sequence revealed >1.8 million canonical EMS-induced G/C to A...
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Analyses of deep mammalian sequence alignments and constraint predictions for 1% of the human genome
Margulies, Elliott H.; Cooper, Gregory M.; Asimenos, George; Thomas, Daryl J.; Dewey, Colin N.; Siepel, Adam; Birney, Ewan; Keefe, Damian; Schwartz, Ariel S.; Hou, Minmei; Taylor, James; Nikolaev, Sergey; Montoya-Burgos, Juan I.; Löytynoja, Ari; Whelan, Simon; Pardi, Fabio; Massingham, Tim; Brown, James B.; Bickel, Peter; Holmes, Ian; Mullikin, James C.; Ureta-Vidal, Abel; Paten, Benedict; Stone, Eric A.; Rosenbloom, Kate R.; Kent, W. James; Bouffard, Gerard G.; Guan, Xiaobin; Hansen, Nancy F.; Idol, Jacquelyn R.; Maduro, Valerie V.B.; Maskeri, Baishali; McDowell, Jennifer C.; Park, Morgan; Thomas, Pamela J.; Young, Alice C.; Blakesley, Robert W.; Muzny, Donna M.; Sodergren, Erica; Wheeler, David A.; Worley, Kim C.; Jiang, Huaiyang; Weinstock, George M.; Gibbs, Richard A.; Graves, Tina; Fulton, Robert; Mardis, Elaine R.; Wilson, Richard K.; Clamp, Michele; Cuff, James; Gnerre, Sante; Jaffe, David B.; Chang, Jean L.; Lindblad-Toh, Kerstin; Lander, Eric S.; Hinrichs, Angie; Trumbower, Heather; Clawson, Hiram; Zweig, Ann; Kuhn, Robert M.; Barber, Galt; Harte, Rachel; Karolchik, Donna; Field, Matthew A.; Moore, Richard A.; Matthewson, Carrie A.; Schein, Jacqueline E.; Marra, Marco A.; Antonarakis, Stylianos E.; Batzoglou, Serafim; Goldman, Nick; Hardison, Ross; Haussler, David; Miller, Webb; Pachter, Lior; Green, Eric D.; Sidow, Arend
2007-01-01
A key component of the ongoing ENCODE project involves rigorous comparative sequence analyses for the initially targeted 1% of the human genome. Here, we present orthologous sequence generation, alignment, and evolutionary constraint analyses of 23 mammalian species for all ENCODE targets. Alignments were generated using four different methods; comparisons of these methods reveal large-scale consistency but substantial differences in terms of small genomic rearrangements, sensitivity (sequence coverage), and specificity (alignment accuracy). We describe the quantitative and qualitative trade-offs concomitant with alignment method choice and the levels of technical error that need to be accounted for in applications that require multisequence alignments. Using the generated alignments, we identified constrained regions using three different methods. While the different constraint-detecting methods are in general agreement, there are important discrepancies relating to both the underlying alignments and the specific algorithms. However, by integrating the results across the alignments and constraint-detecting methods, we produced constraint annotations that were found to be robust based on multiple independent measures. Analyses of these annotations illustrate that most classes of experimentally annotated functional elements are enriched for constrained sequences; however, large portions of each class (with the exception of protein-coding sequences) do not overlap constrained regions. The latter elements might not be under primary sequence constraint, might not be constrained across all mammals, or might have expendable molecular functions. Conversely, 40% of the constrained sequences do not overlap any of the functional elements that have been experimentally identified. Together, these findings demonstrate and quantify how many genomic functional elements await basic molecular characterization. PMID:17567995
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Pauchet, Y; Wilkinson, P; Vogel, H; Nelson, D R; Reynolds, S E; Heckel, D G; ffrench-Constant, R H
2010-02-01
The tobacco hornworm Manduca sexta is an important model for insect physiology but genomic and transcriptomic data are currently lacking. Following a recent pyrosequencing study generating immune related expressed sequence tags (ESTs), here we use this new technology to define the M. sexta larval midgut transcriptome. We generated over 387,000 midgut ESTs, using a combination of Sanger and 454 sequencing, and classified predicted proteins into those involved in digestion, detoxification and immunity. In many cases the depth of 454 pyrosequencing coverage allowed us to define the entire cDNA sequence of a particular gene. Many new M. sexta genes are described including up to 36 new cytochrome P450s, some of which have been implicated in the metabolism of host plant-derived nicotine. New lepidopteran gene families such as the beta-fructofuranosidases, previously thought to be restricted to Bombyx mori, are also described. An unexpectedly high number of ESTs were involved in immunity, for example 39 contigs encoding serpins, and the increasingly appreciated role of the midgut in insect immunity is discussed. Similar studies of other tissues will allow for a tissue by tissue description of the M. sexta transcriptome and will form an essential complimentary step on the road to genome sequencing and annotation.
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Dong, Yan; Zhu, Lixin; Bai, Yu; Ou, Yongyue; Wang, Changbao
2016-01-01
Abstract A lack of mitochondrial genome data from myriapods is hampering progress across genetic, systematic, phylogenetic and evolutionary studies. Here, the complete mitochondrial genomes of two millipedes, Asiomorpha coarctata Saussure, 1860 (Diplopoda: Polydesmida: Paradoxosomatidae) and Xystodesmus sp. (Diplopoda: Polydesmida: Xystodesmidae) were assembled with high coverage using Illumina sequencing data. The mitochondrial genomes of the two newly sequenced species are circular molecules of 15,644 bp and 15,791 bp, within which the typical mitochondrial genome complement of 13 protein-coding genes, 22 tRNAs and two ribosomal RNA genes could be identified. The mitochondrial genome of Asiomorpha coarctata is the first complete sequence in the family Paradoxosomatidae (Diplopoda: Polydesmida) and the gene order of the two flat-backed millipedes is novel among known myriapod mitochondrial genomes. Unique translocations have occurred, including inversion of one half of the two genomes with respect to other millipede genomes. Inversion of the entire side of a genome (trnF-nad5-trnH-nad4-nad4L, trnP, nad1-trnL2-trnL1-rrnL-trnV-rrnS, trnQ, trnC and trnY) could constitute a common event in the order Polydesmida. Last, our phylogenetic analyses recovered the monophyletic Progoneata, subphylum Myriapoda and four internal classes. PMID:28138271
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The draft genome of MD-2 pineapple using hybrid error correction of long reads
Redwan, Raimi M.; Saidin, Akzam; Kumar, S. Vijay
2016-01-01
The introduction of the elite pineapple variety, MD-2, has caused a significant market shift in the pineapple industry. Better productivity, overall increased in fruit quality and taste, resilience to chilled storage and resistance to internal browning are among the key advantages of the MD-2 as compared with its previous predecessor, the Smooth Cayenne. Here, we present the genome sequence of the MD-2 pineapple (Ananas comosus (L.) Merr.) by using the hybrid sequencing technology from two highly reputable platforms, i.e. the PacBio long sequencing reads and the accurate Illumina short reads. Our draft genome achieved 99.6% genome coverage with 27,017 predicted protein-coding genes while 45.21% of the genome was identified as repetitive elements. Furthermore, differential expression of ripening RNASeq library of pineapple fruits revealed ethylene-related transcripts, believed to be involved in regulating the process of non-climacteric pineapple fruit ripening. The MD-2 pineapple draft genome serves as an example of how a complex heterozygous genome is amenable to whole genome sequencing by using a hybrid technology that is both economical and accurate. The genome will make genomic applications more feasible as a medium to understand complex biological processes specific to pineapple. PMID:27374615
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Bringing the fathead minnow (Pimephales promelas) into the ...
The fathead minnow (Pimephales promelas) is a well-established ecotoxicological model organism that has been widely used for regulatory ecotoxicity testing and research for over a half century. Throughout this time, a lot of knowledge has been gained about the fathead minnow’s biological responses to various xenobiotics. However, despite its importance as a model organism, the fathead minnow still has few publicly available gene sequences. Recently, Burns et al. (2015; Environ. Toxicol. Chem. 35:212) described the sequencing and de-novo assembly of the fathead minnow genome. Two draft genome assemblies are now publicly available on the GenBank database. However, on their own the draft assemblies remain of limited use to researchers who are primarily interested in the functional units of the genome, i.e. the genes. In the present study, an annotation pipeline, consisting of gene prediction, evidence alignment, and data synthesis, was applied to the fathead minnow SOAPdenovo assembly. Ab initio gene prediction was performed using AUGUSTUS, which provided a starting point of 43,345 gene predictions. Fathead minnow Expressed Sequence Tags (ESTs) and zebrafish protein-coding sequences (CDSs) were then aligned to the assembly using the corresponding spliced alignment methods of the program Exonerate. Of the over 240,000 EST alignments, 73% were successfully aligned with 90% or greater sequence identity and query coverage. Similarly, 39% of nearly 45,000 zebrafish co
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Assembly of 913 microbial genomes from metagenomic sequencing of the cow rumen.
Stewart, Robert D; Auffret, Marc D; Warr, Amanda; Wiser, Andrew H; Press, Maximilian O; Langford, Kyle W; Liachko, Ivan; Snelling, Timothy J; Dewhurst, Richard J; Walker, Alan W; Roehe, Rainer; Watson, Mick
2018-02-28
The cow rumen is adapted for the breakdown of plant material into energy and nutrients, a task largely performed by enzymes encoded by the rumen microbiome. Here we present 913 draft bacterial and archaeal genomes assembled from over 800 Gb of rumen metagenomic sequence data derived from 43 Scottish cattle, using both metagenomic binning and Hi-C-based proximity-guided assembly. Most of these genomes represent previously unsequenced strains and species. The draft genomes contain over 69,000 proteins predicted to be involved in carbohydrate metabolism, over 90% of which do not have a good match in public databases. Inclusion of the 913 genomes presented here improves metagenomic read classification by sevenfold against our own data, and by fivefold against other publicly available rumen datasets. Thus, our dataset substantially improves the coverage of rumen microbial genomes in the public databases and represents a valuable resource for biomass-degrading enzyme discovery and studies of the rumen microbiome.
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Mapping and analysis of Caenorhabditis elegans transcription factor sequence specificities
Narasimhan, Kamesh; Lambert, Samuel A; Yang, Ally WH; Riddell, Jeremy; Mnaimneh, Sanie; Zheng, Hong; Albu, Mihai; Najafabadi, Hamed S; Reece-Hoyes, John S; Fuxman Bass, Juan I; Walhout, Albertha JM; Weirauch, Matthew T; Hughes, Timothy R
2015-01-01
Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been limited, as DNA-binding motifs are known for only 71 of the estimated 763 sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray experiments on representatives of canonical TF families in C. elegans, obtaining motifs for 129 TFs. Additionally, we predict motifs for many TFs that have DNA-binding domains similar to those already characterized, increasing coverage of binding specificities to 292 C. elegans TFs (∼40%). These data highlight the diversification of binding motifs for the nuclear hormone receptor and C2H2 zinc finger families and reveal unexpected diversity of motifs for T-box and DM families. Motif enrichment in promoters of functionally related genes is consistent with known biology and also identifies putative regulatory roles for unstudied TFs. DOI: http://dx.doi.org/10.7554/eLife.06967.001 PMID:25905672
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NASA Technical Reports Server (NTRS)
1980-01-01
A brief summary of the launch vehicle, spacecraft, and mission is contained. Information relative to launch windows, vehicle telemetry coverage, realtime data flow, telemetry coverage by station, selected trajectory information, and a brief sequence of flight events is also included.
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Phylogenetic appearance of Neuropeptide S precursor proteins in tetrapods
Reinscheid, Rainer K.
2007-01-01
Sleep and emotional behavior are two hallmarks of vertebrate animal behavior, implying that specialized neuronal circuits and dedicated neurochemical messengers may have been developed during evolution to regulate such complex behaviors. Neuropeptide S (NPS) is a newly identified peptide transmitter that activates a typical G protein-coupled receptor. Central administration of NPS produces profound arousal, enhances wakefulness and suppresses all stages of sleep. In addition, NPS can alleviate behavioral responses to stress by producing anxiolytic-like effects. A bioinformatic analysis of current genome databases revealed that the NPS peptide precursor gene is present in all vertebrates with the exception of fish. A high level of sequence conservation, especially of aminoterminal structures was detected, indicating stringent requirements for agonist-induced receptor activation. Duplication of the NPS precursor gene was only found in one out of two marsupial species with sufficient genome coverage (Monodelphis domestica; opossum), indicating that the duplicated opossum NPS sequence might have arisen as an isolated event. Pharmacological analysis of both Monodelphis NPS peptides revealed that only the closely related NPS peptide retained agonistic activity at NPS receptors. The duplicated precursor might be either a pseudogene or could have evolved different receptor selectivity. Together, these data show that NPS is a relatively recent gene in vertebrate evolution whose appearance might coincide with its specialized physiological functions in terrestrial vertebrates. PMID:17293003
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High-Throughput Next-Generation Sequencing of Polioviruses
Montmayeur, Anna M.; Schmidt, Alexander; Zhao, Kun; Magaña, Laura; Iber, Jane; Castro, Christina J.; Chen, Qi; Henderson, Elizabeth; Ramos, Edward; Shaw, Jing; Tatusov, Roman L.; Dybdahl-Sissoko, Naomi; Endegue-Zanga, Marie Claire; Adeniji, Johnson A.; Oberste, M. Steven; Burns, Cara C.
2016-01-01
ABSTRACT The poliovirus (PV) is currently targeted for worldwide eradication and containment. Sanger-based sequencing of the viral protein 1 (VP1) capsid region is currently the standard method for PV surveillance. However, the whole-genome sequence is sometimes needed for higher resolution global surveillance. In this study, we optimized whole-genome sequencing protocols for poliovirus isolates and FTA cards using next-generation sequencing (NGS), aiming for high sequence coverage, efficiency, and throughput. We found that DNase treatment of poliovirus RNA followed by random reverse transcription (RT), amplification, and the use of the Nextera XT DNA library preparation kit produced significantly better results than other preparations. The average viral reads per total reads, a measurement of efficiency, was as high as 84.2% ± 15.6%. PV genomes covering >99 to 100% of the reference length were obtained and validated with Sanger sequencing. A total of 52 PV genomes were generated, multiplexing as many as 64 samples in a single Illumina MiSeq run. This high-throughput, sequence-independent NGS approach facilitated the detection of a diverse range of PVs, especially for those in vaccine-derived polioviruses (VDPV), circulating VDPV, or immunodeficiency-related VDPV. In contrast to results from previous studies on other viruses, our results showed that filtration and nuclease treatment did not discernibly increase the sequencing efficiency of PV isolates. However, DNase treatment after nucleic acid extraction to remove host DNA significantly improved the sequencing results. This NGS method has been successfully implemented to generate PV genomes for molecular epidemiology of the most recent PV isolates. Additionally, the ability to obtain full PV genomes from FTA cards will aid in facilitating global poliovirus surveillance. PMID:27927929
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León, Ileana R.; Schwämmle, Veit; Jensen, Ole N.; Sprenger, Richard R.
2013-01-01
The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods. PMID:23792921
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Zhang, Suhua; Bian, Yingnan; Chen, Anqi; Zheng, Hancheng; Gao, Yuzhen; Hou, Yiping; Li, Chengtao
2017-03-01
Utilizing massively parallel sequencing (MPS) technology for SNP testing in forensic genetics is becoming attractive because of the shortcomings of STR markers, such as their high mutation rates and disadvantages associated with the current PCR-CE method as well as its limitations regarding multiplex capabilities. MPS offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. In this study, we designed a customized SNP panel that includes 273 forensically relevant identity SNPs chosen from SNPforID, IISNP, and the HapMap database as well as previously related studies and evaluated the levels of genotyping precision, sequence coverage, sensitivity and SNP performance using the Ion Torrent PGM. In a concordant study of the custom MPS-SNP panel, only four MPS callings were missing due to coverage reads that were too low (<20), whereas the others were fully concordant with Sanger's sequencing results across the two control samples, that is, 9947A and 9948. The analyses indicated a balanced coverage among the included loci, with the exception of the 16 SNPs that were used to detect an inconsistent allele balance and/or lower coverage reads among 50 tested individuals from the Chinese HAN population and the above controls. With the exception of the 16 poorly performing SNPs, the sequence coverage obtained was extensive for the bulk of the SNPs, and only three Y-SNPs (rs16980601, rs11096432, rs3900) showed a mean coverage below 1000. Analyses of the dilution series of control DNA 9948 yielded reproducible results down to 1ng of DNA input. In addition, we provide an analysis tool for automated data quality control and genotyping checks, and we conclude that the SNP targets are polymorphic and independent in the Chinese HAN population. In summary, the evaluation of the sensitivity, accuracy and genotyping performance provides strong support for the application of MPS technology in forensic SNP analysis, and the assay offers a straightforward sample-to-genotype workflow that could be beneficial in forensic casework with respect to both individual identification and complex kinship issues. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
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Ye, Hongping; Hill, John; Kauffman, John; Gryniewicz, Connie; Han, Xianlin
2008-08-15
Isotope tags for relative and absolute quantification (iTRAQ) reagent coupled with matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometric analysis has been evaluated as both a qualitative and quantitative method for the detection of modifications to active pharmaceutical ingredients derived from recombinant DNA technologies and as a method to detect counterfeit drug products. Five types of insulin (human, bovine, porcine, Lispro, and Lantus) were used as model products in the study because of their minor variations in amino acid sequence. Several experiments were conducted in which each insulin variant was separately digested with Glu-C, and the digestate was labeled with one of four different iTRAQ reagents. All digestates were then combined for desalting and MALDI-TOF/TOF mass spectrometric analysis. When the digestion procedure was optimized, the insulin sequence coverage was 100%. Five different types of insulin were readily differentiated, including human insulin (P28K29) and Lispro insulin (K28P29), which differ only by the interchange of two contiguous residues. Moreover, quantitative analyses show that the results obtained from the iTRAQ method agree well with those determined by other conventional methods. Collectively, the iTRAQ method can be used as a qualitative and quantitative technique for the detection of protein modification and counterfeiting.
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Liu, Wen; Ghouri, Fozia; Yu, Hang; Li, Xiang; Yu, Shuhong; Shahid, Muhammad Qasim; Liu, Xiangdong
2017-01-01
Common wild rice (Oryza rufipogon Griff.) is an important germplasm for rice breeding, which contains many resistance genes. Re-sequencing provides an unprecedented opportunity to explore the abundant useful genes at whole genome level. Here, we identified the nucleotide-binding site leucine-rich repeat (NBS-LRR) encoding genes by re-sequencing of two wild rice lines (i.e. Huaye 1 and Huaye 2) that were developed from common wild rice. We obtained 128 to 147 million reads with approximately 32.5-fold coverage depth, and uniquely covered more than 89.6% (> = 1 fold) of reference genomes. Two wild rice lines showed high SNP (single-nucleotide polymorphisms) variation rate in 12 chromosomes against the reference genomes of Nipponbare (japonica cultivar) and 93-11 (indica cultivar). InDels (insertion/deletion polymorphisms) count-length distribution exhibited normal distribution in the two lines, and most of the InDels were ranged from -5 to 5 bp. With reference to the Nipponbare genome sequence, we detected a total of 1,209,308 SNPs, 161,117 InDels and 4,192 SVs (structural variations) in Huaye 1, and 1,387,959 SNPs, 180,226 InDels and 5,305 SVs in Huaye 2. A total of 44.9% and 46.9% genes exhibited sequence variations in two wild rice lines compared to the Nipponbare and 93-11 reference genomes, respectively. Analysis of NBS-LRR mutant candidate genes showed that they were mainly distributed on chromosome 11, and NBS domain was more conserved than LRR domain in both wild rice lines. NBS genes depicted higher levels of genetic diversity in Huaye 1 than that found in Huaye 2. Furthermore, protein-protein interaction analysis showed that NBS genes mostly interacted with the cytochrome C protein (Os05g0420600, Os01g0885000 and BGIOSGA038922), while some NBS genes interacted with heat shock protein, DNA-binding activity, Phosphoinositide 3-kinase and a coiled coil region. We explored abundant NBS-LRR encoding genes in two common wild rice lines through genome wide re-sequencing, which proved to be a useful tool to exploit elite NBS-LRR genes in wild rice. The data here provide a foundation for future work aimed at dissecting the genetic basis of disease resistance in rice, and the two wild rice lines will be useful germplasm for the molecular improvement of cultivated rice.
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Yu, Hang; Li, Xiang; Yu, Shuhong; Shahid, Muhammad Qasim
2017-01-01
Common wild rice (Oryza rufipogon Griff.) is an important germplasm for rice breeding, which contains many resistance genes. Re-sequencing provides an unprecedented opportunity to explore the abundant useful genes at whole genome level. Here, we identified the nucleotide-binding site leucine-rich repeat (NBS-LRR) encoding genes by re-sequencing of two wild rice lines (i.e. Huaye 1 and Huaye 2) that were developed from common wild rice. We obtained 128 to 147 million reads with approximately 32.5-fold coverage depth, and uniquely covered more than 89.6% (> = 1 fold) of reference genomes. Two wild rice lines showed high SNP (single-nucleotide polymorphisms) variation rate in 12 chromosomes against the reference genomes of Nipponbare (japonica cultivar) and 93–11 (indica cultivar). InDels (insertion/deletion polymorphisms) count-length distribution exhibited normal distribution in the two lines, and most of the InDels were ranged from -5 to 5 bp. With reference to the Nipponbare genome sequence, we detected a total of 1,209,308 SNPs, 161,117 InDels and 4,192 SVs (structural variations) in Huaye 1, and 1,387,959 SNPs, 180,226 InDels and 5,305 SVs in Huaye 2. A total of 44.9% and 46.9% genes exhibited sequence variations in two wild rice lines compared to the Nipponbare and 93–11 reference genomes, respectively. Analysis of NBS-LRR mutant candidate genes showed that they were mainly distributed on chromosome 11, and NBS domain was more conserved than LRR domain in both wild rice lines. NBS genes depicted higher levels of genetic diversity in Huaye 1 than that found in Huaye 2. Furthermore, protein-protein interaction analysis showed that NBS genes mostly interacted with the cytochrome C protein (Os05g0420600, Os01g0885000 and BGIOSGA038922), while some NBS genes interacted with heat shock protein, DNA-binding activity, Phosphoinositide 3-kinase and a coiled coil region. We explored abundant NBS-LRR encoding genes in two common wild rice lines through genome wide re-sequencing, which proved to be a useful tool to exploit elite NBS-LRR genes in wild rice. The data here provide a foundation for future work aimed at dissecting the genetic basis of disease resistance in rice, and the two wild rice lines will be useful germplasm for the molecular improvement of cultivated rice. PMID:28700714
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Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits.
Imprialou, Martha; Kahles, André; Steffen, Joshua G; Osborne, Edward J; Gan, Xiangchao; Lempe, Janne; Bhomra, Amarjit; Belfield, Eric; Visscher, Anne; Greenhalgh, Robert; Harberd, Nicholas P; Goram, Richard; Hein, Jotun; Robert-Seilaniantz, Alexandre; Jones, Jonathan; Stegle, Oliver; Kover, Paula; Tsiantis, Miltos; Nordborg, Magnus; Rätsch, Gunnar; Clark, Richard M; Mott, Richard
2017-04-01
To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii , isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions. Copyright © 2017 by the Genetics Society of America.
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Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.
2013-01-01
Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423
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Kirkwood, Kathryn J; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I
2013-12-01
Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.
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Ancestry estimation and control of population stratification for sequence-based association studies.
Wang, Chaolong; Zhan, Xiaowei; Bragg-Gresham, Jennifer; Kang, Hyun Min; Stambolian, Dwight; Chew, Emily Y; Branham, Kari E; Heckenlively, John; Fulton, Robert; Wilson, Richard K; Mardis, Elaine R; Lin, Xihong; Swaroop, Anand; Zöllner, Sebastian; Abecasis, Gonçalo R
2014-04-01
Estimating individual ancestry is important in genetic association studies where population structure leads to false positive signals, although assigning ancestry remains challenging with targeted sequence data. We propose a new method for the accurate estimation of individual genetic ancestry, based on direct analysis of off-target sequence reads, and implement our method in the publicly available LASER software. We validate the method using simulated and empirical data and show that the method can accurately infer worldwide continental ancestry when used with sequencing data sets with whole-genome shotgun coverage as low as 0.001×. For estimates of fine-scale ancestry within Europe, the method performs well with coverage of 0.1×. On an even finer scale, the method improves discrimination between exome-sequenced study participants originating from different provinces within Finland. Finally, we show that our method can be used to improve case-control matching in genetic association studies and to reduce the risk of spurious findings due to population structure.
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Large-scale label-free quantitative proteomics of the pea aphid-Buchnera symbiosis.
Poliakov, Anton; Russell, Calum W; Ponnala, Lalit; Hoops, Harold J; Sun, Qi; Douglas, Angela E; van Wijk, Klaas J
2011-06-01
Many insects are nutritionally dependent on symbiotic microorganisms that have tiny genomes and are housed in specialized host cells called bacteriocytes. The obligate symbiosis between the pea aphid Acyrthosiphon pisum and the γ-proteobacterium Buchnera aphidicola (only 584 predicted proteins) is particularly amenable for molecular analysis because the genomes of both partners have been sequenced. To better define the symbiotic relationship between this aphid and Buchnera, we used large-scale, high accuracy tandem mass spectrometry (nanoLC-LTQ-Orbtrap) to identify aphid and Buchnera proteins in the whole aphid body, purified bacteriocytes, isolated Buchnera cells and the residual bacteriocyte fraction. More than 1900 aphid and 400 Buchnera proteins were identified. All enzymes in amino acid metabolism annotated in the Buchnera genome were detected, reflecting the high (68%) coverage of the proteome and supporting the core function of Buchnera in the aphid symbiosis. Transporters mediating the transport of predicted metabolites were present in the bacteriocyte. Label-free spectral counting combined with hierarchical clustering, allowed to define the quantitative distribution of a subset of these proteins across both symbiotic partners, yielding no evidence for the selective transfer of protein among the partners in either direction. This is the first quantitative proteome analysis of bacteriocyte symbiosis, providing a wealth of information about molecular function of both the host cell and bacterial symbiont.
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Recovering complete and draft population genomes from metagenome datasets
Sangwan, Naseer; Xia, Fangfang; Gilbert, Jack A.
2016-03-08
Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem ofmore » chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution.« less
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Recovering complete and draft population genomes from metagenome datasets
DOE Office of Scientific and Technical Information (OSTI.GOV)
Sangwan, Naseer; Xia, Fangfang; Gilbert, Jack A.
Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem ofmore » chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution.« less
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Artieri, Carlo G; Fraser, Hunter B
2014-12-01
The recent advent of ribosome profiling-sequencing of short ribosome-bound fragments of mRNA-has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling data set have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a method that implicated positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that it produces false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling. © 2014 Artieri and Fraser; Published by Cold Spring Harbor Laboratory Press.
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New in-depth rainbow trout transcriptome reference and digital atlas of gene expression
USDA-ARS?s Scientific Manuscript database
Sequencing the rainbow trout genome is underway and a transcriptome reference sequence is required to help in genome assembly and gene discovery. Previously, we reported a transcriptome reference sequence using a 19X coverage of 454-pyrosequencing data. Although this work added a great wealth of ann...
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USDA-ARS?s Scientific Manuscript database
Genomic structural variations are an important source of genetic diversity. Copy number variations (CNVs), gains and losses of large regions of genomic sequence between individuals of a species, are known to be associated with both diseases and phenotypic traits. Deeply sequenced genomes are often u...
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Suckau, Detlev; Resemann, Anja
2009-12-01
The ability to match Top-Down protein sequencing (TDS) results by MALDI-TOF to protein sequences by classical protein database searching was evaluated in this work. Resulting from these analyses were the protein identity, the simultaneous assignment of the N- and C-termini and protein sequences of up to 70 residues from either terminus. In combination with de novo sequencing using the MALDI-TDS data, even fusion proteins were assigned and the detailed sequence around the fusion site was elucidated. MALDI-TDS allowed to efficiently match protein sequences quickly and to validate recombinant protein structures-in particular, protein termini-on the level of undigested proteins.
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Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.
Adalsteinsson, Viktor A; Ha, Gavin; Freeman, Samuel S; Choudhury, Atish D; Stover, Daniel G; Parsons, Heather A; Gydush, Gregory; Reed, Sarah C; Rotem, Denisse; Rhoades, Justin; Loginov, Denis; Livitz, Dimitri; Rosebrock, Daniel; Leshchiner, Ignaty; Kim, Jaegil; Stewart, Chip; Rosenberg, Mara; Francis, Joshua M; Zhang, Cheng-Zhong; Cohen, Ofir; Oh, Coyin; Ding, Huiming; Polak, Paz; Lloyd, Max; Mahmud, Sairah; Helvie, Karla; Merrill, Margaret S; Santiago, Rebecca A; O'Connor, Edward P; Jeong, Seong H; Leeson, Rachel; Barry, Rachel M; Kramkowski, Joseph F; Zhang, Zhenwei; Polacek, Laura; Lohr, Jens G; Schleicher, Molly; Lipscomb, Emily; Saltzman, Andrea; Oliver, Nelly M; Marini, Lori; Waks, Adrienne G; Harshman, Lauren C; Tolaney, Sara M; Van Allen, Eliezer M; Winer, Eric P; Lin, Nancy U; Nakabayashi, Mari; Taplin, Mary-Ellen; Johannessen, Cory M; Garraway, Levi A; Golub, Todd R; Boehm, Jesse S; Wagle, Nikhil; Getz, Gad; Love, J Christopher; Meyerson, Matthew
2017-11-06
Whole-exome sequencing of cell-free DNA (cfDNA) could enable comprehensive profiling of tumors from blood but the genome-wide concordance between cfDNA and tumor biopsies is uncertain. Here we report ichorCNA, software that quantifies tumor content in cfDNA from 0.1× coverage whole-genome sequencing data without prior knowledge of tumor mutations. We apply ichorCNA to 1439 blood samples from 520 patients with metastatic prostate or breast cancers. In the earliest tested sample for each patient, 34% of patients have ≥10% tumor-derived cfDNA, sufficient for standard coverage whole-exome sequencing. Using whole-exome sequencing, we validate the concordance of clonal somatic mutations (88%), copy number alterations (80%), mutational signatures, and neoantigens between cfDNA and matched tumor biopsies from 41 patients with ≥10% cfDNA tumor content. In summary, we provide methods to identify patients eligible for comprehensive cfDNA profiling, revealing its applicability to many patients, and demonstrate high concordance of cfDNA and metastatic tumor whole-exome sequencing.
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The study of human Y chromosome variation through ancient DNA.
Kivisild, Toomas
2017-05-01
High throughput sequencing methods have completely transformed the study of human Y chromosome variation by offering a genome-scale view on genetic variation retrieved from ancient human remains in context of a growing number of high coverage whole Y chromosome sequence data from living populations from across the world. The ancient Y chromosome sequences are providing us the first exciting glimpses into the past variation of male-specific compartment of the genome and the opportunity to evaluate models based on previously made inferences from patterns of genetic variation in living populations. Analyses of the ancient Y chromosome sequences are challenging not only because of issues generally related to ancient DNA work, such as DNA damage-induced mutations and low content of endogenous DNA in most human remains, but also because of specific properties of the Y chromosome, such as its highly repetitive nature and high homology with the X chromosome. Shotgun sequencing of uniquely mapping regions of the Y chromosomes to sufficiently high coverage is still challenging and costly in poorly preserved samples. To increase the coverage of specific target SNPs capture-based methods have been developed and used in recent years to generate Y chromosome sequence data from hundreds of prehistoric skeletal remains. Besides the prospects of testing directly as how much genetic change in a given time period has accompanied changes in material culture the sequencing of ancient Y chromosomes allows us also to better understand the rate at which mutations accumulate and get fixed over time. This review considers genome-scale evidence on ancient Y chromosome diversity that has recently started to accumulate in geographic areas favourable to DNA preservation. More specifically the review focuses on examples of regional continuity and change of the Y chromosome haplogroups in North Eurasia and in the New World.
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Kalra, Shikha; Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Kumar, Sunil; Kaur, Jagdeep; Ramachandran, Srinivasan; Singh, Kashmir
2013-01-01
Chlorophytum borivilianum, an endangered medicinal plant species is highly recognized for its aphrodisiac properties provided by saponins present in the plant. The transcriptome information of this species is limited and only few hundred expressed sequence tags (ESTs) are available in the public databases. To gain molecular insight of this plant, high throughput transcriptome sequencing of leaf RNA was carried out using Illumina's HiSeq 2000 sequencing platform. A total of 22,161,444 single end reads were retrieved after quality filtering. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 101,141 assembled transcripts were obtained, with coverage size of 22.42 Mb and average length of 221 bp. Guanine-cytosine (GC) content was found to be 44%. Bioinformatics analysis, using non-redundant proteins, gene ontology (GO), enzyme commission (EC) and kyoto encyclopedia of genes and genomes (KEGG) databases, extracted all the known enzymes involved in saponin and flavonoid biosynthesis. Few genes of the alkaloid biosynthesis, along with anticancer and plant defense genes, were also discovered. Additionally, several cytochrome P450 (CYP450) and glycosyltransferase unique sequences were also found. We identified simple sequence repeat motifs in transcripts with an abundance of di-nucleotide simple sequence repeat (SSR; 43.1%) markers. Large scale expression profiling through Reads per Kilobase per Million mapped reads (RPKM) showed major genes involved in different metabolic pathways of the plant. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of C. borivilianum. PMID:24376689
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Improve homology search sensitivity of PacBio data by correcting frameshifts.
Du, Nan; Sun, Yanni
2016-09-01
Single-molecule, real-time sequencing (SMRT) developed by Pacific BioSciences produces longer reads than secondary generation sequencing technologies such as Illumina. The long read length enables PacBio sequencing to close gaps in genome assembly, reveal structural variations, and identify gene isoforms with higher accuracy in transcriptomic sequencing. However, PacBio data has high sequencing error rate and most of the errors are insertion or deletion errors. During alignment-based homology search, insertion or deletion errors in genes will cause frameshifts and may only lead to marginal alignment scores and short alignments. As a result, it is hard to distinguish true alignments from random alignments and the ambiguity will incur errors in structural and functional annotation. Existing frameshift correction tools are designed for data with much lower error rate and are not optimized for PacBio data. As an increasing number of groups are using SMRT, there is an urgent need for dedicated homology search tools for PacBio data. In this work, we introduce Frame-Pro, a profile homology search tool for PacBio reads. Our tool corrects sequencing errors and also outputs the profile alignments of the corrected sequences against characterized protein families. We applied our tool to both simulated and real PacBio data. The results showed that our method enables more sensitive homology search, especially for PacBio data sets of low sequencing coverage. In addition, we can correct more errors when comparing with a popular error correction tool that does not rely on hybrid sequencing. The source code is freely available at https://sourceforge.net/projects/frame-pro/ yannisun@msu.edu. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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Kalra, Shikha; Puniya, Bhanwar Lal; Kulshreshtha, Deepika; Kumar, Sunil; Kaur, Jagdeep; Ramachandran, Srinivasan; Singh, Kashmir
2013-01-01
Chlorophytum borivilianum, an endangered medicinal plant species is highly recognized for its aphrodisiac properties provided by saponins present in the plant. The transcriptome information of this species is limited and only few hundred expressed sequence tags (ESTs) are available in the public databases. To gain molecular insight of this plant, high throughput transcriptome sequencing of leaf RNA was carried out using Illumina's HiSeq 2000 sequencing platform. A total of 22,161,444 single end reads were retrieved after quality filtering. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 101,141 assembled transcripts were obtained, with coverage size of 22.42 Mb and average length of 221 bp. Guanine-cytosine (GC) content was found to be 44%. Bioinformatics analysis, using non-redundant proteins, gene ontology (GO), enzyme commission (EC) and kyoto encyclopedia of genes and genomes (KEGG) databases, extracted all the known enzymes involved in saponin and flavonoid biosynthesis. Few genes of the alkaloid biosynthesis, along with anticancer and plant defense genes, were also discovered. Additionally, several cytochrome P450 (CYP450) and glycosyltransferase unique sequences were also found. We identified simple sequence repeat motifs in transcripts with an abundance of di-nucleotide simple sequence repeat (SSR; 43.1%) markers. Large scale expression profiling through Reads per Kilobase per Million mapped reads (RPKM) showed major genes involved in different metabolic pathways of the plant. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of C. borivilianum.
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Wahjuhono, Gendro; Revolusiana; Widhiastuti, Dyah; Sundoro, Julitasari; Mardani, Tri; Ratih, Woro Umi; Sutomo, Retno; Safitri, Ida; Sampurno, Ondri Dwi; Rana, Bardan; Roivainen, Merja; Kahn, Anna-Lea; Mach, Ondrej; Pallansch, Mark A; Sutter, Roland W
2014-11-01
Inactivated poliovirus vaccine (IPV) is rarely used in tropical developing countries. To generate additional scientific information, especially on the possible emergence of vaccine-derived polioviruses (VDPVs) in an IPV-only environment, we initiated an IPV introduction project in Yogyakarta, an Indonesian province. In this report, we present the coverage, immunity, and VDPV surveillance results. In Yogyakarta, we established environmental surveillance starting in 2004; and conducted routine immunization coverage and seroprevalence surveys before and after a September 2007 switch from oral poliovirus vaccine (OPV) to IPV, using standard coverage and serosurvey methods. Rates and types of polioviruses found in sewage samples were analyzed, and all poliovirus isolates after the switch were sequenced. Vaccination coverage (>95%) and immunity (approximately 100%) did not change substantially before and after the IPV switch. No VDPVs were detected. Before the switch, 58% of environmental samples contained Sabin poliovirus; starting 6 weeks after the switch, Sabin polioviruses were rarely isolated, and if they were, genetic sequencing suggested recent introductions. This project demonstrated that under almost ideal conditions (good hygiene, maintenance of universally high IPV coverage, and corresponding high immunity against polioviruses), no emergence and circulation of VDPV could be detected in a tropical developing country setting. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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Hücker, Sarah M.; Ardern, Zachary; Goldberg, Tatyana; Schafferhans, Andrea; Bernhofer, Michael; Vestergaard, Gisle; Nelson, Chase W.; Schloter, Michael; Rost, Burkhard; Scherer, Siegfried
2017-01-01
In the past, short protein-coding genes were often disregarded by genome annotation pipelines. Transcriptome sequencing (RNAseq) signals outside of annotated genes have usually been interpreted to indicate either ncRNA or pervasive transcription. Therefore, in addition to the transcriptome, the translatome (RIBOseq) of the enteric pathogen Escherichia coli O157:H7 strain Sakai was determined at two optimal growth conditions and a severe stress condition combining low temperature and high osmotic pressure. All intergenic open reading frames potentially encoding a protein of ≥ 30 amino acids were investigated with regard to coverage by transcription and translation signals and their translatability expressed by the ribosomal coverage value. This led to discovery of 465 unique, putative novel genes not yet annotated in this E. coli strain, which are evenly distributed over both DNA strands of the genome. For 255 of the novel genes, annotated homologs in other bacteria were found, and a machine-learning algorithm, trained on small protein-coding E. coli genes, predicted that 89% of these translated open reading frames represent bona fide genes. The remaining 210 putative novel genes without annotated homologs were compared to the 255 novel genes with homologs and to 250 short annotated genes of this E. coli strain. All three groups turned out to be similar with respect to their translatability distribution, fractions of differentially regulated genes, secondary structure composition, and the distribution of evolutionary constraint, suggesting that both novel groups represent legitimate genes. However, the machine-learning algorithm only recognized a small fraction of the 210 genes without annotated homologs. It is possible that these genes represent a novel group of genes, which have unusual features dissimilar to the genes of the machine-learning algorithm training set. PMID:28902868
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Flexible CRISPR library construction using parallel oligonucleotide retrieval
Read, Abigail; Gao, Shaojian; Batchelor, Eric
2017-01-01
Abstract CRISPR/Cas9-based gene knockout libraries have emerged as a powerful tool for functional screens. We present here a set of pre-designed human and mouse sgRNA sequences that are optimized for both high on-target potency and low off-target effect. To maximize the chance of target gene inactivation, sgRNAs were curated to target both 5΄ constitutive exons and exons that encode conserved protein domains. We describe here a robust and cost-effective method to construct multiple small sized CRISPR library from a single oligo pool generated by array synthesis using parallel oligonucleotide retrieval. Together, these resources provide a convenient means for individual labs to generate customized CRISPR libraries of variable size and coverage depth for functional genomics application. PMID:28334828
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Comparative genomic data of the Avian Phylogenomics Project.
Zhang, Guojie; Li, Bo; Li, Cai; Gilbert, M Thomas P; Jarvis, Erich D; Wang, Jun
2014-01-01
The evolutionary relationships of modern birds are among the most challenging to understand in systematic biology and have been debated for centuries. To address this challenge, we assembled or collected the genomes of 48 avian species spanning most orders of birds, including all Neognathae and two of the five Palaeognathae orders, and used the genomes to construct a genome-scale avian phylogenetic tree and perform comparative genomics analyses (Jarvis et al. in press; Zhang et al. in press). Here we release assemblies and datasets associated with the comparative genome analyses, which include 38 newly sequenced avian genomes plus previously released or simultaneously released genomes of Chicken, Zebra finch, Turkey, Pigeon, Peregrine falcon, Duck, Budgerigar, Adelie penguin, Emperor penguin and the Medium Ground Finch. We hope that this resource will serve future efforts in phylogenomics and comparative genomics. The 38 bird genomes were sequenced using the Illumina HiSeq 2000 platform and assembled using a whole genome shotgun strategy. The 48 genomes were categorized into two groups according to the N50 scaffold size of the assemblies: a high depth group comprising 23 species sequenced at high coverage (>50X) with multiple insert size libraries resulting in N50 scaffold sizes greater than 1 Mb (except the White-throated Tinamou and Bald Eagle); and a low depth group comprising 25 species sequenced at a low coverage (~30X) with two insert size libraries resulting in an average N50 scaffold size of about 50 kb. Repetitive elements comprised 4%-22% of the bird genomes. The assembled scaffolds allowed the homology-based annotation of 13,000 ~ 17000 protein coding genes in each avian genome relative to chicken, zebra finch and human, as well as comparative and sequence conservation analyses. Here we release full genome assemblies of 38 newly sequenced avian species, link genome assembly downloads for the 7 of the remaining 10 species, and provide a guideline of genomic data that has been generated and used in our Avian Phylogenomics Project. To the best of our knowledge, the Avian Phylogenomics Project is the biggest vertebrate comparative genomics project to date. The genomic data presented here is expected to accelerate further analyses in many fields, including phylogenetics, comparative genomics, evolution, neurobiology, development biology, and other related areas.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Shi, CY; Yang, H; Wei, CL
Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Using high-throughput Illumina RNA-seq, the transcriptome from poly (A){sup +} RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled intomore » 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR). An extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis.« less
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2011-01-01
Background Tea is one of the most popular non-alcoholic beverages worldwide. However, the tea plant, Camellia sinensis, is difficult to culture in vitro, to transform, and has a large genome, rendering little genomic information available. Recent advances in large-scale RNA sequencing (RNA-seq) provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes. Results Using high-throughput Illumina RNA-seq, the transcriptome from poly (A)+ RNA of C. sinensis was analyzed at an unprecedented depth (2.59 gigabase pairs). Approximate 34.5 million reads were obtained, trimmed, and assembled into 127,094 unigenes, with an average length of 355 bp and an N50 of 506 bp, which consisted of 788 contig clusters and 126,306 singletons. This number of unigenes was 10-fold higher than existing C. sinensis sequences deposited in GenBank (as of August 2010). Sequence similarity analyses against six public databases (Uniprot, NR and COGs at NCBI, Pfam, InterPro and KEGG) found 55,088 unigenes that could be annotated with gene descriptions, conserved protein domains, or gene ontology terms. Some of the unigenes were assigned to putative metabolic pathways. Targeted searches using these annotations identified the majority of genes associated with several primary metabolic pathways and natural product pathways that are important to tea quality, such as flavonoid, theanine and caffeine biosynthesis pathways. Novel candidate genes of these secondary pathways were discovered. Comparisons with four previously prepared cDNA libraries revealed that this transcriptome dataset has both a high degree of consistency with previous EST data and an approximate 20 times increase in coverage. Thirteen unigenes related to theanine and flavonoid synthesis were validated. Their expression patterns in different organs of the tea plant were analyzed by RT-PCR and quantitative real time PCR (qRT-PCR). Conclusions An extensive transcriptome dataset has been obtained from the deep sequencing of tea plant. The coverage of the transcriptome is comprehensive enough to discover all known genes of several major metabolic pathways. This transcriptome dataset can serve as an important public information platform for gene expression, genomics, and functional genomic studies in C. sinensis. PMID:21356090
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Exome sequencing for prenatal diagnosis of fetuses with sonographic abnormalities.
Drury, Suzanne; Williams, Hywel; Trump, Natalie; Boustred, Christopher; Lench, Nicholas; Scott, Richard H; Chitty, Lyn S
2015-10-01
In the absence of aneuploidy or other pathogenic cytogenetic abnormality, fetuses with increased nuchal translucency (NT ≥ 3.5 mm) and/or other sonographic abnormalities have a greater incidence of genetic syndromes, but defining the underlying pathology can be challenging. Here, we investigate the value of whole exome sequencing in fetuses with sonographic abnormalities but normal microarray analysis. Whole exome sequencing was performed on DNA extracted from chorionic villi or amniocytes in 24 fetuses with unexplained ultrasound findings. In the first 14 cases sequencing was initially performed on fetal DNA only. For the remaining 10, the trio of fetus, mother and father was sequenced simultaneously. In 21% (5/24) cases, exome sequencing provided definitive diagnoses (Milroy disease, hypophosphatasia, achondrogenesis type 2, Freeman-Sheldon syndrome and Baraitser-Winter Syndrome). In a further case, a plausible diagnosis of orofaciodigital syndrome type 6 was made. In two others, a single mutation in an autosomal recessive gene was identified, but incomplete sequencing coverage precluded exclusion of the presence of a second mutation. Whole exome sequencing improves prenatal diagnosis in euploid fetuses with abnormal ultrasound scans. In order to expedite interpretation of results, trio sequencing should be employed, but interpretation can still be compromised by incomplete coverage of relevant genes. © 2015 John Wiley & Sons, Ltd.
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Leese, Florian; Mayer, Christoph; Agrawal, Shobhit; Dambach, Johannes; Dietz, Lars; Doemel, Jana S.; Goodall-Copstake, William P.; Held, Christoph; Jackson, Jennifer A.; Lampert, Kathrin P.; Linse, Katrin; Macher, Jan N.; Nolzen, Jennifer; Raupach, Michael J.; Rivera, Nicole T.; Schubart, Christoph D.; Striewski, Sebastian; Tollrian, Ralph; Sands, Chester J.
2012-01-01
High throughput sequencing technologies are revolutionizing genetic research. With this “rise of the machines”, genomic sequences can be obtained even for unknown genomes within a short time and for reasonable costs. This has enabled evolutionary biologists studying genetically unexplored species to identify molecular markers or genomic regions of interest (e.g. micro- and minisatellites, mitochondrial and nuclear genes) by sequencing only a fraction of the genome. However, when using such datasets from non-model species, it is possible that DNA from non-target contaminant species such as bacteria, viruses, fungi, or other eukaryotic organisms may complicate the interpretation of the results. In this study we analysed 14 genomic pyrosequencing libraries of aquatic non-model taxa from four major evolutionary lineages. We quantified the amount of suitable micro- and minisatellites, mitochondrial genomes, known nuclear genes and transposable elements and searched for contamination from various sources using bioinformatic approaches. Our results show that in all sequence libraries with estimated coverage of about 0.02–25%, many appropriate micro- and minisatellites, mitochondrial gene sequences and nuclear genes from different KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways could be identified and characterized. These can serve as markers for phylogenetic and population genetic analyses. A central finding of our study is that several genomic libraries suffered from different biases owing to non-target DNA or mobile elements. In particular, viruses, bacteria or eukaryote endosymbionts contributed significantly (up to 10%) to some of the libraries analysed. If not identified as such, genetic markers developed from high-throughput sequencing data for non-model organisms may bias evolutionary studies or fail completely in experimental tests. In conclusion, our study demonstrates the enormous potential of low-coverage genome survey sequences and suggests bioinformatic analysis workflows. The results also advise a more sophisticated filtering for problematic sequences and non-target genome sequences prior to developing markers. PMID:23185309
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Verifying Digital Components of Physical Systems: Experimental Evaluation of Test Quality
NASA Astrophysics Data System (ADS)
Laputenko, A. V.; López, J. E.; Yevtushenko, N. V.
2018-03-01
This paper continues the study of high quality test derivation for verifying digital components which are used in various physical systems; those are sensors, data transfer components, etc. We have used logic circuits b01-b010 of the package of ITC'99 benchmarks (Second Release) for experimental evaluation which as stated before, describe digital components of physical systems designed for various applications. Test sequences are derived for detecting the most known faults of the reference logic circuit using three different approaches to test derivation. Three widely used fault types such as stuck-at-faults, bridges, and faults which slightly modify the behavior of one gate are considered as possible faults of the reference behavior. The most interesting test sequences are short test sequences that can provide appropriate guarantees after testing, and thus, we experimentally study various approaches to the derivation of the so-called complete test suites which detect all fault types. In the first series of experiments, we compare two approaches for deriving complete test suites. In the first approach, a shortest test sequence is derived for testing each fault. In the second approach, a test sequence is pseudo-randomly generated by the use of an appropriate software for logic synthesis and verification (ABC system in our study) and thus, can be longer. However, after deleting sequences detecting the same set of faults, a test suite returned by the second approach is shorter. The latter underlines the fact that in many cases it is useless to spend `time and efforts' for deriving a shortest distinguishing sequence; it is better to use the test minimization afterwards. The performed experiments also show that the use of only randomly generated test sequences is not very efficient since such sequences do not detect all the faults of any type. After reaching the fault coverage around 70%, saturation is observed, and the fault coverage cannot be increased anymore. For deriving high quality short test suites, the approach that is the combination of randomly generated sequences together with sequences which are aimed to detect faults not detected by random tests, allows to reach the good fault coverage using shortest test sequences.
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Improved multiple displacement amplification (iMDA) and ultraclean reagents.
Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W
2014-06-06
Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.
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Nguyen, Nam-Ninh; Srihari, Sriganesh; Leong, Hon Wai; Chong, Ket-Fah
2015-10-01
Determining the entire complement of enzymes and their enzymatic functions is a fundamental step for reconstructing the metabolic network of cells. High quality enzyme annotation helps in enhancing metabolic networks reconstructed from the genome, especially by reducing gaps and increasing the enzyme coverage. Currently, structure-based and network-based approaches can only cover a limited number of enzyme families, and the accuracy of homology-based approaches can be further improved. Bottom-up homology-based approach improves the coverage by rebuilding Hidden Markov Model (HMM) profiles for all known enzymes. However, its clustering procedure relies firmly on BLAST similarity score, ignoring protein domains/patterns, and is sensitive to changes in cut-off thresholds. Here, we use functional domain architecture to score the association between domain families and enzyme families (Domain-Enzyme Association Scoring, DEAS). The DEAS score is used to calculate the similarity between proteins, which is then used in clustering procedure, instead of using sequence similarity score. We improve the enzyme annotation protocol using a stringent classification procedure, and by choosing optimal threshold settings and checking for active sites. Our analysis shows that our stringent protocol EnzDP can cover up to 90% of enzyme families available in Swiss-Prot. It achieves a high accuracy of 94.5% based on five-fold cross-validation. EnzDP outperforms existing methods across several testing scenarios. Thus, EnzDP serves as a reliable automated tool for enzyme annotation and metabolic network reconstruction. Available at: www.comp.nus.edu.sg/~nguyennn/EnzDP .
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Towards Discovery and Targeted Peptide Biomarker Detection Using nanoESI-TIMS-TOF MS
NASA Astrophysics Data System (ADS)
Garabedian, Alyssa; Benigni, Paolo; Ramirez, Cesar E.; Baker, Erin S.; Liu, Tao; Smith, Richard D.; Fernandez-Lima, Francisco
2018-05-01
In the present work, the potential of trapped ion mobility spectrometry coupled to TOF mass spectrometry (TIMS-TOF MS) for discovery and targeted monitoring of peptide biomarkers from human-in-mouse xenograft tumor tissue was evaluated. In particular, a TIMS-MS workflow was developed for the detection and quantification of peptide biomarkers using internal heavy analogs, taking advantage of the high mobility resolution (R = 150-250) prior to mass analysis. Five peptide biomarkers were separated, identified, and quantified using offline nanoESI-TIMS-CID-TOF MS; the results were in good agreement with measurements using a traditional LC-ESI-MS/MS proteomics workflow. The TIMS-TOF MS analysis permitted peptide biomarker detection based on accurate mobility, mass measurements, and high sequence coverage for concentrations in the 10-200 nM range, while simultaneously achieving discovery measurements of not initially targeted peptides as markers from the same proteins and, eventually, other proteins. [Figure not available: see fulltext.
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Heidler, Juliana; Hardt, Stefanie; Wittig, Ilka; Tegeder, Irmgard
2016-12-01
Progranulin deficiency is associated with neurodegeneration in humans and in mice. The mechanisms likely involve progranulin-promoted removal of protein waste via autophagy. We performed a deep proteomic screen of the pre-frontal cortex in aged (13-15 months) female progranulin-deficient mice (GRN -/- ) and mice with inducible neuron-specific overexpression of progranulin (SLICK-GRN-OE) versus the respective control mice. Proteins were extracted and analyzed per liquid chromatography/mass spectrometry (LC/MS) on a Thermo Scientific™ Q Exactive Plus equipped with an ultra-high performance liquid chromatography unit and a Nanospray Flex Ion-Source. Full Scan MS-data were acquired using Xcalibur and raw files were analyzed using the proteomics software Max Quant. The mouse reference proteome set from uniprot (June 2015) was used to identify peptides and proteins. The DiB data file is a reduced MaxQuant output and includes peptide and protein identification, accession numbers, protein and gene names, sequence coverage and label free quantification (LFQ) values of each sample. Differences in protein expression in genotypes are presented in "Progranulin overexpression in sensory neurons attenuates neuropathic pain in mice: Role of autophagy" (C. Altmann, S. Hardt, C. Fischer, J. Heidler, H.Y. Lim, A. Haussler, B. Albuquerque, B. Zimmer, C. Moser, C. Behrends, F. Koentgen, I. Wittig, M.H. Schmidt, A.M. Clement, T. Deller, I. Tegeder, 2016) [1].
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Zhang, Xi
2016-12-01
Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins-at high efficiency and peptide reproducibility-poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
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Alam, Syed Imteyaz; Dwivedi, Pratistha
2016-10-01
The whole genome sequencing and annotation of Clostridium perfringens strains revealed several genes coding for proteins of unknown function with no significant similarities to genes in other organisms. Our previous studies clearly demonstrated that hypothetical proteins CPF_2500, CPF_1441, CPF_0876, CPF_0093, CPF_2002, CPF_2314, CPF_1179, CPF_1132, CPF_2853, CPF_0552, CPF_2032, CPF_0438, CPF_1440, CPF_2918, CPF_0656, and CPF_2364 are genuine proteins of C. perfringens expressed in high abundance. This study explored the putative role of these hypothetical proteins using bioinformatic tools and evaluated their potential as putative candidates for prophylaxis. Apart from a group of eight hypothetical proteins (HPs), a putative function was predicted for the rest of the hypothetical proteins using one or more of the algorithms used. The phylogenetic analysis did not suggest an evidence of a horizontal gene transfer event except for HP CPF_0876. HP CPF_2918 is an abundant extracellular protein, unique to C. perfringens species with maximum strain coverage and did not show any significant match in the database. CPF_2918 was cloned, recombinant protein was purified to near homogeneity, and probing with mouse anti-CPF_2918 serum revealed surface localization of the protein in C. perfringens ATCC13124 cultures. The purified recombinant CPF_2918 protein induced antibody production, a mixed Th1 and Th2 kind of response, and provided partial protection to immunized mice in direct C. perfringens challenge. Copyright © 2016 Elsevier B.V. All rights reserved.
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Green, Luke R; Lucidarme, Jay; Dave, Neelam; Chan, Hannah; Clark, Stephen; Borrow, Ray; Bayliss, Christopher D
2018-06-27
A recombinant NadA protein is one of the four major protective antigens of 4C-MenB (Bexsero®), a vaccine developed for serogroup B Neisseria meningitidis (MenB). The Meningococcal Antigen Typing System (MATS) is utilised as a high throughput assay for assessing the invasive MenB strain coverage of 4C-MenB. Where present, the nadA gene is subject to phase variable changes in transcription due to a 5'TAAA repeat tract located in a regulatory region. The promoter-containing intergenic region sequences (IGR) and 5'TAAA repeat numbers were determined for 906 invasive meningococcal disease isolates possessing the nadA gene. Exclusion of the 5'TAAA repeats reduced the number of IGR alleles from 82 to 23. Repeat numbers were associated with low and high levels of NadA expression by Western blotting and ELISA. Low expression repeat numbers were present in 83% of 179 MenB isolates with NadA-2/3 or Nad-1 peptide variants and 68% of 480 MenW ST-11 complex isolates with Nad-2/3 peptide variants. For isolates with vaccine-compatible NadA variants, 93% of MATS negative isolates were associated with low expression repeat numbers whereas 63% of isolates with MATS RP scores above the 95% confidence interval for the positive bactericidal threshold had high expression repeat numbers. Analysis of the 5'TAAA repeat number has potential as a rapid, high throughput method for assessing strain coverage for the NadA-component of 4C-MenB. A key application will be assessing coverage in meningococcal disease cases where confirmation is by PCR only and MATS cannot be applied. Copyright © 2018 Green et al.
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Mikkelsen, Martin; Frank-Hansen, Rune; Hansen, Anders J; Morling, Niels
2014-09-01
of sequencing of whole mitochondrial genome, HV1 and HV2 DNA with the second generation system (SGS) Roche 454 GS Junior were compared with results of Sanger sequencing and SNP typing with SNaPshot single base extension detected with MALDI-TOF and capillary electrophoresis. We investigated the performance of the software analysis of the data, reproducibility, ability to sequence homopolymeric regions, detection of mixtures and heteroplasmy as well as the implications of the depth of coverage. We found full reproducibility between samples sequenced twice with SGS. We found close to full concordance between the mtDNA sequences of 26 samples obtained with (1) the 454 SGS method using a depth of coverage above 100 and (2) Sanger sequencing and SNP typing. The discrepancies were primarily observed in homopolymeric regions. The 454 SGS method was able to sequence 95% of the reads correctly in homopolymers up to 4 bases, and up to 6 bases could be sequenced with similar success if the results were carefully, visually inspected. The 454 technology was able to detect mixtures or heteroplasmy of approximately 10%. We detected previously unreported heteroplasmy in the GM9947A component of the NIST human mitochondrial DNA SRM-2392 standard reference material. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.
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Biedrzycka, Aleksandra; Sebastian, Alvaro; Migalska, Magdalena; Westerdahl, Helena; Radwan, Jacek
2017-07-01
Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage. © 2016 John Wiley & Sons Ltd.
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AD-LIBS: inferring ancestry across hybrid genomes using low-coverage sequence data.
Schaefer, Nathan K; Shapiro, Beth; Green, Richard E
2017-04-04
Inferring the ancestry of each region of admixed individuals' genomes is useful in studies ranging from disease gene mapping to speciation genetics. Current methods require high-coverage genotype data and phased reference panels, and are therefore inappropriate for many data sets. We present a software application, AD-LIBS, that uses a hidden Markov model to infer ancestry across hybrid genomes without requiring variant calling or phasing. This approach is useful for non-model organisms and in cases of low-coverage data, such as ancient DNA. We demonstrate the utility of AD-LIBS with synthetic data. We then use AD-LIBS to infer ancestry in two published data sets: European human genomes with Neanderthal ancestry and brown bear genomes with polar bear ancestry. AD-LIBS correctly infers 87-91% of ancestry in simulations and produces ancestry maps that agree with published results and global ancestry estimates in humans. In brown bears, we find more polar bear ancestry than has been published previously, using both AD-LIBS and an existing software application for local ancestry inference, HAPMIX. We validate AD-LIBS polar bear ancestry maps by recovering a geographic signal within bears that mirrors what is seen in SNP data. Finally, we demonstrate that AD-LIBS is more effective than HAPMIX at inferring ancestry when preexisting phased reference data are unavailable and genomes are sequenced to low coverage. AD-LIBS is an effective tool for ancestry inference that can be used even when few individuals are available for comparison or when genomes are sequenced to low coverage. AD-LIBS is therefore likely to be useful in studies of non-model or ancient organisms that lack large amounts of genomic DNA. AD-LIBS can therefore expand the range of studies in which admixture mapping is a viable tool.
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Insurance coverage of medical foods for treatment of inherited metabolic disorders
Berry, Susan A.; Kenney, Mary Kay; Harris, Katharine B.; Singh, Rani H.; Cameron, Cynthia A.; Kraszewski, Jennifer N.; Levy-Fisch, Jill; Shuger, Jill F.; Greene, Carol L.; Lloyd-Puryear, Michele A.; Boyle, Coleen A.
2015-01-01
Purpose Treatment of inherited metabolic disorders is accomplished by use of specialized diets employing medical foods and medically necessary supplements. Families seeking insurance coverage for these products express concern that coverage is often limited; the extent of this challenge is not well defined. Methods To learn about limitations in insurance coverage, parents of 305 children with inherited metabolic disorders completed a paper survey providing information about their use of medical foods, modified low-protein foods, prescribed dietary supplements, and medical feeding equipment and supplies for treatment of their child's disorder as well as details about payment sources for these products. Results Although nearly all children with inherited metabolic dis orders had medical coverage of some type, families paid “out of pocket” for all types of products. Uncovered spending was reported for 11% of families purchasing medical foods, 26% purchasing supplements, 33% of those needing medical feeding supplies, and 59% of families requiring modified low-protein foods. Forty-two percent of families using modified low-protein foods and 21% of families using medical foods reported additional treatment-related expenses of $100 or more per month for these products. Conclusion Costs of medical foods used to treat inherited metabolic disorders are not completely covered by insurance or other resources. PMID:23598714
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Serrano, Pedro; Dutta, Samit K; Proudfoot, Andrew; Mohanty, Biswaranjan; Susac, Lukas; Martin, Bryan; Geralt, Michael; Jaroszewski, Lukasz; Godzik, Adam; Elsliger, Marc; Wilson, Ian A; Wüthrich, Kurt
2016-11-01
For more than a decade, the Joint Center for Structural Genomics (JCSG; www.jcsg.org) worked toward increased three-dimensional structure coverage of the protein universe. This coordinated quest was one of the main goals of the four high-throughput (HT) structure determination centers of the Protein Structure Initiative (PSI; www.nigms.nih.gov/Research/specificareas/PSI). To achieve the goals of the PSI, the JCSG made use of the complementarity of structure determination by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy to increase and diversify the range of targets entering the HT structure determination pipeline. The overall strategy, for both techniques, was to determine atomic resolution structures for representatives of large protein families, as defined by the Pfam database, which had no structural coverage and could make significant contributions to biological and biomedical research. Furthermore, the experimental structures could be leveraged by homology modeling to further expand the structural coverage of the protein universe and increase biological insights. Here, we describe what could be achieved by this structural genomics approach, using as an illustration the contributions from 20 NMR structure determinations out of a total of 98 JCSG NMR structures, which were selected because they are the first three-dimensional structure representations of the respective Pfam protein families. The information from this small sample is representative for the overall results from crystal and NMR structure determination in the JCSG. There are five new folds, which were classified as domains of unknown functions (DUF), three of the proteins could be functionally annotated based on three-dimensional structure similarity with previously characterized proteins, and 12 proteins showed only limited similarity with previous deposits in the Protein Data Bank (PDB) and were classified as DUFs. © 2016 Federation of European Biochemical Societies.
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Algorithm, applications and evaluation for protein comparison by Ramanujan Fourier transform.
Zhao, Jian; Wang, Jiasong; Hua, Wei; Ouyang, Pingkai
2015-12-01
The amino acid sequence of a protein determines its chemical properties, chain conformation and biological functions. Protein sequence comparison is of great importance to identify similarities of protein structures and infer their functions. Many properties of a protein correspond to the low-frequency signals within the sequence. Low frequency modes in protein sequences are linked to the secondary structures, membrane protein types, and sub-cellular localizations of the proteins. In this paper, we present Ramanujan Fourier transform (RFT) with a fast algorithm to analyze the low-frequency signals of protein sequences. The RFT method is applied to similarity analysis of protein sequences with the Resonant Recognition Model (RRM). The results show that the proposed fast RFT method on protein comparison is more efficient than commonly used discrete Fourier transform (DFT). RFT can detect common frequencies as significant feature for specific protein families, and the RFT spectrum heat-map of protein sequences demonstrates the information conservation in the sequence comparison. The proposed method offers a new tool for pattern recognition, feature extraction and structural analysis on protein sequences. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Beer, Lynn A; Wang, Huan; Tang, Hsin-Yao; Cao, Zhijun; Chang-Wong, Tony; Tanyi, Janos L; Zhang, Rugang; Liu, Qin; Speicher, David W
2013-01-01
The most cancer-specific biomarkers in blood are likely to be proteins shed directly by the tumor rather than less specific inflammatory or other host responses. The use of xenograft mouse models together with in-depth proteome analysis for identification of human proteins in the mouse blood is an under-utilized strategy that can clearly identify proteins shed by the tumor. In the current study, 268 human proteins shed into mouse blood from human OVCAR-3 serous tumors were identified based upon human vs. mouse species differences using a four-dimensional plasma proteome fractionation strategy. A multi-step prioritization and verification strategy was subsequently developed to efficiently select some of the most promising biomarkers from this large number of candidates. A key step was parallel analysis of human proteins detected in the tumor supernatant, because substantially greater sequence coverage for many of the human proteins initially detected in the xenograft mouse plasma confirmed assignments as tumor-derived human proteins. Verification of candidate biomarkers in patient sera was facilitated by in-depth, label-free quantitative comparisons of serum pools from patients with ovarian cancer and benign ovarian tumors. The only proteins that advanced to multiple reaction monitoring (MRM) assay development were those that exhibited increases in ovarian cancer patients compared with benign tumor controls. MRM assays were facilely developed for all 11 novel biomarker candidates selected by this process and analysis of larger pools of patient sera suggested that all 11 proteins are promising candidate biomarkers that should be further evaluated on individual patient blood samples.
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Kammermeier, Jochen; Drury, Suzanne; James, Chela T; Dziubak, Robert; Ocaka, Louise; Elawad, Mamoun; Beales, Philip; Lench, Nicholas; Uhlig, Holm H; Bacchelli, Chiara; Shah, Neil
2014-11-01
Multiple monogenetic conditions with partially overlapping phenotypes can present with inflammatory bowel disease (IBD)-like intestinal inflammation. With novel genotype-specific therapies emerging, establishing a molecular diagnosis is becoming increasingly important. We have introduced targeted next-generation sequencing (NGS) technology as a prospective screening tool in children with very early onset IBD (VEOIBD). We evaluated the coverage of 40 VEOIBD genes in two separate cohorts undergoing targeted gene panel sequencing (TGPS) (n=25) and whole exome sequencing (WES) (n=20). TGPS revealed causative mutations in four genes (IL10RA, EPCAM, TTC37 and SKIV2L) discovered unexpected phenotypes and directly influenced clinical decision making by supporting as well as avoiding haematopoietic stem cell transplantation. TGPS resulted in significantly higher median coverage when compared with WES, fewer coverage deficiencies and improved variant detection across established VEOIBD genes. Excluding or confirming known VEOIBD genotypes should be considered early in the disease course in all cases of therapy-refractory VEOIBD, as it can have a direct impact on patient management. To combine both described NGS technologies would compensate for the limitations of WES for disease-specific application while offering the opportunity for novel gene discovery in the research setting. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
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Folding and Stabilization of Native-Sequence-Reversed Proteins
Zhang, Yuanzhao; Weber, Jeffrey K; Zhou, Ruhong
2016-01-01
Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols. PMID:27113844
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Folding and Stabilization of Native-Sequence-Reversed Proteins
NASA Astrophysics Data System (ADS)
Zhang, Yuanzhao; Weber, Jeffrey K.; Zhou, Ruhong
2016-04-01
Though the problem of sequence-reversed protein folding is largely unexplored, one might speculate that reversed native protein sequences should be significantly more foldable than purely random heteropolymer sequences. In this article, we investigate how the reverse-sequences of native proteins might fold by examining a series of small proteins of increasing structural complexity (α-helix, β-hairpin, α-helix bundle, and α/β-protein). Employing a tandem protein structure prediction algorithmic and molecular dynamics simulation approach, we find that the ability of reverse sequences to adopt native-like folds is strongly influenced by protein size and the flexibility of the native hydrophobic core. For β-hairpins with reverse-sequences that fail to fold, we employ a simple mutational strategy for guiding stable hairpin formation that involves the insertion of amino acids into the β-turn region. This systematic look at reverse sequence duality sheds new light on the problem of protein sequence-structure mapping and may serve to inspire new protein design and protein structure prediction protocols.
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Freschi de Barros, Samar; De Amicis, Karine Marafigo; Alencar, Raquel; Smeesters, Pierre Robert; Trunkel, Ariel; Postól, Edilberto; Almeida Junior, João Nóbrega; Rossi, Flavia; Pignatari, Antonio Carlos Campos; Kalil, Jorge; Guilherme, Luiza
2015-08-05
Several human diseases are caused by Streptococcus pyogenes, ranging from common infections to autoimmunity. Characterization of the most prevalent strains worldwide is a useful tool for evaluating the coverage capacity of vaccines under development. In this study, a collection of S. pyogenes strains from Sao Paulo, Brazil, was analyzed to describe the diversity of strains and assess the vaccine coverage capacity of StreptInCor. Molecular epidemiology of S. pyogenes strains was performed by emm-genotyping the 229 isolates from different clinical sites, and PCR was used for superantigen profile analysis. The emm-pattern and tissue tropism for these M types were also predicted and compared based on the emm-cluster classification. The strains were fit into 12 different emm-clusters, revealing a diverse phylogenetic origin and, consequently, different mechanisms of infection and escape of the host immune system. Forty-eight emm-types were distinguished in 229 samples, and the 10 most frequently observed types accounted for 69 % of all isolates, indicating a diverse profile of circulating strains comparable to other countries under development. A similar proportion of E and A-C emm-patterns were observed, whereas pattern D was less frequent, indicating that the strains of this collection primarily had a tissue tropism for the throat. In silico analysis of the coverage capacity of StreptInCor, an M protein-conserved regionally based vaccine candidate developed by our group, had a range of 94.5 % to 59.7 %, with a mean of 71.0 % identity between the vaccine antigen and the predicted amino acid sequence of the emm-types included here. This is the first report of S. pyogenes strain characterization in Sao Paulo, one of the largest cities in the world; thus, the strain panel described here is a representative sample for vaccine coverage capacity analysis. Our results enabled evaluation of StreptInCor candidate vaccine coverage capacity against diverse M-types, indicating that the vaccine candidate likely would induce protection against the diverse strains worldwide.
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A HIGH COVERAGE GENOME SEQUENCE FROM AN ARCHAIC DENISOVAN INDIVIDUAL
Meyer, Matthias; Kircher, Martin; Gansauge, Marie-Theres; Li, Heng; Racimo, Fernando; Mallick, Swapan; Schraiber, Joshua G.; Jay, Flora; Prüfer, Kay; de Filippo, Cesare; Sudmant, Peter H.; Alkan, Can; Fu, Qiaomei; Do, Ron; Rohland, Nadin; Tandon, Arti; Siebauer, Michael; Green, Richard E.; Bryc, Katarzyna; Briggs, Adrian W.; Stenzel, Udo; Dabney, Jesse; Shendure, Jay; Kitzman, Jacob; Hammer, Michael F.; Shunkov, Michael V.; Derevianko, Anatoli P.; Patterson, Nick; Andrés, Aida M.; Eichler, Evan E.; Slatkin, Montgomery; Reich, David; Kelso, Janet; Pääbo, Svante
2013-01-01
We present a DNA library preparation method that has allowed us to reconstruct a high coverage (30X) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans. PMID:22936568
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Elisa, Mwega; Hasan, Salih Dia; Moses, Njahira; Elpidius, Rukambile; Skilton, Robert; Gwakisa, Paul
2015-04-01
This study investigated the genetic and antigenic diversity of Theileria parva in cattle from the Eastern and Southern zones of Tanzania. Thirty-nine (62%) positive samples were genotyped using 14 mini- and microsatellite markers with coverage of all four T. parva chromosomes. Wright's F index (F(ST) = 0 × 094) indicated a high level of panmixis. Linkage equilibrium was observed in the two zones studied, suggesting existence of a panmyctic population. In addition, sequence analysis of CD8+ T-cell target antigen genes Tp1 revealed a single protein sequence in all samples analysed, which is also present in the T. parva Muguga strain, which is a component of the FAO1 vaccine. All Tp2 epitope sequences were identical to those in the T. parva Muguga strain, except for one variant of a Tp2 epitope, which is found in T. parva Kiambu 5 strain, also a component the FAO1 vaccine. Neighbour joining tree of the nucleotide sequences of Tp2 showed clustering according to geographical origin. Our results show low genetic and antigenic diversity of T. parva within the populations analysed. This has very important implications for the development of sustainable control measures for T. parva in Eastern and Southern zones of Tanzania, where East Coast fever is endemic.
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The Genome Sequence of a Widespread Apex Predator, the Golden Eagle (Aquila chrysaetos)
Doyle, Jacqueline M.; Katzner, Todd E.; Bloom, Peter H.; Ji, Yanzhu; Wijayawardena, Bhagya K.; DeWoody, J. Andrew
2014-01-01
Biologists routinely use molecular markers to identify conservation units, to quantify genetic connectivity, to estimate population sizes, and to identify targets of selection. Many imperiled eagle populations require such efforts and would benefit from enhanced genomic resources. We sequenced, assembled, and annotated the first eagle genome using DNA from a male golden eagle (Aquila chrysaetos) captured in western North America. We constructed genomic libraries that were sequenced using Illumina technology and assembled the high-quality data to a depth of ∼40x coverage. The genome assembly includes 2,552 scaffolds >10 Kb and 415 scaffolds >1.2 Mb. We annotated 16,571 genes that are involved in myriad biological processes, including such disparate traits as beak formation and color vision. We also identified repetitive regions spanning 92 Mb (∼6% of the assembly), including LINES, SINES, LTR-RTs and DNA transposons. The mitochondrial genome encompasses 17,332 bp and is ∼91% identical to the Mountain Hawk-Eagle (Nisaetus nipalensis). Finally, the data reveal that several anonymous microsatellites commonly used for population studies are embedded within protein-coding genes and thus may not have evolved in a neutral fashion. Because the genome sequence includes ∼800,000 novel polymorphisms, markers can now be chosen based on their proximity to functional genes involved in migration, carnivory, and other biological processes. PMID:24759626
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Laffitte, Marie-Claude N.; Leprohon, Philippe; Hainse, Maripier; Légaré, Danielle; Masson, Jean-Yves; Ouellette, Marc
2016-01-01
The parasite Leishmania often relies on gene rearrangements to survive stressful environments. However, safeguarding a minimum level of genome integrity is important for cell survival. We hypothesized that maintenance of genomic integrity in Leishmania would imply a leading role of the MRE11 and RAD50 proteins considering their role in DNA repair, chromosomal organization and protection of chromosomes ends in other organisms. Attempts to generate RAD50 null mutants in a wild-type background failed and we provide evidence that this gene is essential. Remarkably, inactivation of RAD50 was possible in a MRE11 null mutant that we had previously generated, providing good evidence that RAD50 may be dispensable in the absence of MRE11. Inactivation of the MRE11 and RAD50 genes led to a decreased frequency of homologous recombination and analysis of the null mutants by whole genome sequencing revealed several chromosomal translocations. Sequencing of the junction between translocated chromosomes highlighted microhomology sequences at the level of breakpoint regions. Sequencing data also showed a decreased coverage at subtelomeric locations in many chromosomes in the MRE11-/-RAD50-/- parasites. This study demonstrates an MRE11-independent microhomology-mediated end-joining mechanism and a prominent role for MRE11 and RAD50 in the maintenance of genomic integrity. Moreover, we suggest the possible involvement of RAD50 in subtelomeric regions stability. PMID:27314941
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Circular codes revisited: a statistical approach.
Gonzalez, D L; Giannerini, S; Rosa, R
2011-04-21
In 1996 Arquès and Michel [1996. A complementary circular code in the protein coding genes. J. Theor. Biol. 182, 45-58] discovered the existence of a common circular code in eukaryote and prokaryote genomes. Since then, circular code theory has provoked great interest and underwent a rapid development. In this paper we discuss some theoretical issues related to the synchronization properties of coding sequences and circular codes with particular emphasis on the problem of retrieval and maintenance of the reading frame. Motivated by the theoretical discussion, we adopt a rigorous statistical approach in order to try to answer different questions. First, we investigate the covering capability of the whole class of 216 self-complementary, C(3) maximal codes with respect to a large set of coding sequences. The results indicate that, on average, the code proposed by Arquès and Michel has the best covering capability but, still, there exists a great variability among sequences. Second, we focus on such code and explore the role played by the proportion of the bases by means of a hierarchy of permutation tests. The results show the existence of a sort of optimization mechanism such that coding sequences are tailored as to maximize or minimize the coverage of circular codes on specific reading frames. Such optimization clearly relates the function of circular codes with reading frame synchronization. Copyright © 2011 Elsevier Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Borot de Battisti, M; Maenhout, M; Lagendijk, J J W
Purpose: To develop a new method which adaptively determines the optimal needle insertion sequence for HDR prostate brachytherapy involving divergent needle-by-needle dose delivery by e.g. a robotic device. A needle insertion sequence is calculated at the beginning of the intervention and updated after each needle insertion with feedback on needle positioning errors. Methods: Needle positioning errors and anatomy changes may occur during HDR brachytherapy which can lead to errors in the delivered dose. A novel strategy was developed to calculate and update the needle sequence and the dose plan after each needle insertion with feedback on needle positioning errors. Themore » dose plan optimization was performed by numerical simulations. The proposed needle sequence determination optimizes the final dose distribution based on the dose coverage impact of each needle. This impact is predicted stochastically by needle insertion simulations. HDR procedures were simulated with varying number of needle insertions (4 to 12) using 11 patient MR data-sets with PTV, prostate, urethra, bladder and rectum delineated. Needle positioning errors were modeled by random normally distributed angulation errors (standard deviation of 3 mm at the needle’s tip). The final dose parameters were compared in the situations where the needle with the largest vs. the smallest dose coverage impact was selected at each insertion. Results: Over all scenarios, the percentage of clinically acceptable final dose distribution improved when the needle selected had the largest dose coverage impact (91%) compared to the smallest (88%). The differences were larger for few (4 to 6) needle insertions (maximum difference scenario: 79% vs. 60%). The computation time of the needle sequence optimization was below 60s. Conclusion: A new adaptive needle sequence determination for HDR prostate brachytherapy was developed. Coupled to adaptive planning, the selection of the needle with the largest dose coverage impact increases chances of reaching the clinical constraints. M. Borot de Battisti is funded by Philips Medical Systems Nederland B.V.; M. Moerland is principal investigator on a contract funded by Philips Medical Systems Nederland B.V.; G. Hautvast and D. Binnekamp are fulltime employees of Philips Medical Systems Nederland B.V.« less
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Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys.
Barouch, Dan H; O'Brien, Kara L; Simmons, Nathaniel L; King, Sharon L; Abbink, Peter; Maxfield, Lori F; Sun, Ying-Hua; La Porte, Annalena; Riggs, Ambryice M; Lynch, Diana M; Clark, Sarah L; Backus, Katherine; Perry, James R; Seaman, Michael S; Carville, Angela; Mansfield, Keith G; Szinger, James J; Fischer, Will; Muldoon, Mark; Korber, Bette
2010-03-01
The worldwide diversity of HIV-1 presents an unprecedented challenge for vaccine development. Antigens derived from natural HIV-1 sequences have elicited only a limited breadth of cellular immune responses in nonhuman primate studies and clinical trials to date. Polyvalent 'mosaic' antigens, in contrast, are designed to optimize cellular immunologic coverage of global HIV-1 sequence diversity. Here we show that mosaic HIV-1 Gag, Pol and Env antigens expressed by recombinant, replication-incompetent adenovirus serotype 26 vectors markedly augmented both the breadth and depth without compromising the magnitude of antigen-specific T lymphocyte responses as compared with consensus or natural sequence HIV-1 antigens in rhesus monkeys. Polyvalent mosaic antigens therefore represent a promising strategy to expand cellular immunologic vaccine coverage for genetically diverse pathogens such as HIV-1.
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AGeNNT: annotation of enzyme families by means of refined neighborhood networks.
Kandlinger, Florian; Plach, Maximilian G; Merkl, Rainer
2017-05-25
Large enzyme families may contain functionally diverse members that give rise to clusters in a sequence similarity network (SSN). In prokaryotes, the genome neighborhood of a gene-product is indicative of its function and thus, a genome neighborhood network (GNN) deduced for an SSN provides strong clues to the specific function of enzymes constituting the different clusters. The Enzyme Function Initiative ( http://enzymefunction.org/ ) offers services that compute SSNs and GNNs. We have implemented AGeNNT that utilizes these services, albeit with datasets purged with respect to unspecific protein functions and overrepresented species. AGeNNT generates refined GNNs (rGNNs) that consist of cluster-nodes representing the sequences under study and Pfam-nodes representing enzyme functions encoded in the respective neighborhoods. For cluster-nodes, AGeNNT summarizes the phylogenetic relationships of the contributing species and a statistic indicates how unique nodes and GNs are within this rGNN. Pfam-nodes are annotated with additional features like GO terms describing protein function. For edges, the coverage is given, which is the relative number of neighborhoods containing the considered enzyme function (Pfam-node). AGeNNT is available at https://github.com/kandlinf/agennt . An rGNN is easier to interpret than a conventional GNN, which commonly contains proteins without enzymatic function and overly specific neighborhoods due to phylogenetic bias. The implemented filter routines and the statistic allow the user to identify those neighborhoods that are most indicative of a specific metabolic capacity. Thus, AGeNNT facilitates to distinguish and annotate functionally different members of enzyme families.
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Zhang, Xi
2016-01-01
Neurotransmitter ligand-gated ion channels (LGICs) are widespread and pivotal in brain functions. Unveiling their structure-function mechanisms is crucial to drive drug discovery, and demands robust proteomic quantitation of expression, post-translational modifications (PTMs) and dynamic structures. Yet unbiased digestion of these modified transmembrane proteins—at high efficiency and peptide reproducibility—poses the obstacle. Targeting both enzyme-substrate contacts and PTMs for peptide formation and detection, we devised flow-and-detergent-facilitated protease and de-PTM digestions for deep sequencing (FDD) method that combined omni-compatible detergent, tandem immobilized protease/PNGase columns, and Cys-selective reduction/alkylation, to achieve streamlined ultradeep peptide preparation within minutes not days, at high peptide reproducibility and low abundance-bias. FDD transformed enzyme-protein contacts into equal catalytic travel paths through enzyme-excessive columns regardless of protein abundance, removed products instantly preventing inhibition, tackled intricate structures via sequential multiple micro-digestions along the flow, and precisely controlled peptide formation by flow rate. Peptide-stage reactions reduced steric bias; low contamination deepened MS/MS scan; distinguishing disulfide from M oxidation and avoiding gain/loss artifacts unmasked protein-endogenous oxidation states. Using a recent interactome of 285-kDa human GABA type A receptor, this pilot study validated FDD platform's applicability to deep sequencing (up to 99% coverage), H/D-exchange and TMT-based structural mapping. FDD discovered novel subunit-specific PTM signatures, including unusual nontop-surface N-glycosylations, that may drive subunit biases in human Cys-loop LGIC assembly and pharmacology, by redefining subunit/ligand interfaces and connecting function domains. PMID:27073180
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Miura, Naoki; Kucho, Ken-Ichi; Noguchi, Michiko; Miyoshi, Noriaki; Uchiumi, Toshiki; Kawaguchi, Hiroaki; Tanimoto, Akihide
2014-01-01
The microminipig, which weighs less than 10 kg at an early stage of maturity, has been reported as a potential experimental model animal. Its extremely small size and other distinct characteristics suggest the possibility of a number of differences between the genome of the microminipig and that of conventional pigs. In this study, we analyzed the genomes of two healthy microminipigs using a next-generation sequencer SOLiD™ system. We then compared the obtained genomic sequences with a genomic database for the domestic pig (Sus scrofa). The mapping coverage of sequenced tag from the microminipig to conventional pig genomic sequences was greater than 96% and we detected no clear, substantial genomic variance from these data. The results may indicate that the distinct characteristics of the microminipig derive from small-scale alterations in the genome, such as Single Nucleotide Polymorphisms or translational modifications, rather than large-scale deletion or insertion polymorphisms. Further investigation of the entire genomic sequence of the microminipig with methods enabling deeper coverage is required to elucidate the genetic basis of its distinct phenotypic traits. Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.
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Isolation and Analysis of Keratins and Keratin-Associated Proteins from Hair and Wool.
Deb-Choudhury, Santanu; Plowman, Jeffrey E; Harland, Duane P
2016-01-01
The presence of highly cross-linked protein networks in hair and wool makes them very difficult substrates for protein extraction, a prerequisite for further protein analysis and characterization. It is therefore imperative that these cross-links formed by disulfide bridges are first disrupted for the efficient extraction of proteins. Chaotropes such as urea are commonly used as efficient extractants. However, a combination of urea and thiourea not only improves recovery of proteins but also results in improved resolution of the keratins in 2DE gels. Reductants also play an important role in protein dissolution. Dithiothreitol effectively removes keratinous material from the cortex, whereas phosphines, like Tris(2-carboxyethyl)phosphine, remove material from the exocuticle. The relative extractability of the keratins and keratin-associated proteins is also dependent on the concentration of chaotropes, reductants, and pH, thus providing a means to preferentially extract these proteins. Ionic liquids such as 1-butyl-3-methylimidazolium chloride (BMIM(+)[Cl](-)) are known to solubilize wool by disrupting noncovalent interactions, specifically intermolecular hydrogen bonds. BMIM(+)[Cl](-) proved to be an effective extractant of wool proteins and complementary in nature to chaotropes such as urea and thiourea for identifying unique peptides of wool proteins using mass spectrometry (MS). Successful identification of proteins resolved by one- or two-dimensional electrophoresis and MS is highly dependent on the optimal recovery of its protease-digested peptides with an efficient removal of interfering substances. The detergent sodium deoxycholate used in conjunction with Empore™ disks improved identification of proteins by mass spectrometry leading to higher percentage sequence coverage, identification of unique peptides and higher score. Copyright © 2016 Elsevier Inc. All rights reserved.
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Mass spectrometric determination of early and advanced glycation in biology.
Rabbani, Naila; Ashour, Amal; Thornalley, Paul J
2016-08-01
Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1-5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N(ε)-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them - amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine - particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.
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Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry.
Planatscher, Hannes; Supper, Jochen; Poetz, Oliver; Stoll, Dieter; Joos, Thomas; Templin, Markus F; Zell, Andreas
2010-06-25
Mass spectrometry (MS) based protein profiling has become one of the key technologies in biomedical research and biomarker discovery. One bottleneck in MS-based protein analysis is sample preparation and an efficient fractionation step to reduce the complexity of the biological samples, which are too complex to be analyzed directly with MS. Sample preparation strategies that reduce the complexity of tryptic digests by using immunoaffinity based methods have shown to lead to a substantial increase in throughput and sensitivity in the proteomic mass spectrometry approach. The limitation of using such immunoaffinity-based approaches is the availability of the appropriate peptide specific capture antibodies. Recent developments in these approaches, where subsets of peptides with short identical terminal sequences can be enriched using antibodies directed against short terminal epitopes, promise a significant gain in efficiency. We show that the minimal set of terminal epitopes for the coverage of a target protein list can be found by the formulation as a set cover problem, preceded by a filtering pipeline for the exclusion of peptides and target epitopes with undesirable properties. For small datasets (a few hundred proteins) it is possible to solve the problem to optimality with moderate computational effort using commercial or free solvers. Larger datasets, like full proteomes require the use of heuristics.
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Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin
NASA Astrophysics Data System (ADS)
Gobom, Johan; Nordhoff, Eckhard; Ekman, Rolf; Roepstorff, Peter
1997-12-01
In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversedphase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.
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Comparison and quantitative verification of mapping algorithms for whole genome bisulfite sequencing
USDA-ARS?s Scientific Manuscript database
Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitat...
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Fast imputation using medium or low-coverage sequence data
USDA-ARS?s Scientific Manuscript database
Accurate genotype imputation can greatly reduce costs and increase benefits by combining whole-genome sequence data of varying read depth and microarray genotypes of varying densities. For large populations, an efficient strategy chooses the two haplotypes most likely to form each genotype and updat...
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Role of Mitochondrial Inheritance on Prostate Cancer Outcome in African American Men. Addendum
2016-11-01
DNA sequencing technique developed by our collaborator using single amplicon long-range PCR that permits deep coverage (10,000-20,000X on average) of...the mitochondrial genome. We have sequenced 652 samples derived from frozen fully using this technology. The additional DNA samples derived from...paraffin embedded (FFPE) tissue were more challenging, but have now been sequenced . Mapping of DNA variants in our sequenced genomes to mitochondrial
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Harris, R. Alan; Wang, Ting; Coarfa, Cristian; Nagarajan, Raman P.; Hong, Chibo; Downey, Sara L.; Johnson, Brett E.; Fouse, Shaun D.; Delaney, Allen; Zhao, Yongjun; Olshen, Adam; Ballinger, Tracy; Zhou, Xin; Forsberg, Kevin J.; Gu, Junchen; Echipare, Lorigail; O’Geen, Henriette; Lister, Ryan; Pelizzola, Mattia; Xi, Yuanxin; Epstein, Charles B.; Bernstein, Bradley E.; Hawkins, R. David; Ren, Bing; Chung, Wen-Yu; Gu, Hongcang; Bock, Christoph; Gnirke, Andreas; Zhang, Michael Q.; Haussler, David; Ecker, Joseph; Li, Wei; Farnham, Peggy J.; Waterland, Robert A.; Meissner, Alexander; Marra, Marco A.; Hirst, Martin; Milosavljevic, Aleksandar; Costello, Joseph F.
2010-01-01
Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression. PMID:20852635
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NASA Astrophysics Data System (ADS)
Lindner, Robert; Lou, Xinghua; Reinstein, Jochen; Shoeman, Robert L.; Hamprecht, Fred A.; Winkler, Andreas
2014-06-01
Hydrogen-deuterium exchange (HDX) experiments analyzed by mass spectrometry (MS) provide information about the dynamics and the solvent accessibility of protein backbone amide hydrogen atoms. Continuous improvement of MS instrumentation has contributed to the increasing popularity of this method; however, comprehensive automated data analysis is only beginning to mature. We present Hexicon 2, an automated pipeline for data analysis and visualization based on the previously published program Hexicon (Lou et al. 2010). Hexicon 2 employs the sensitive NITPICK peak detection algorithm of its predecessor in a divide-and-conquer strategy and adds new features, such as chromatogram alignment and improved peptide sequence assignment. The unique feature of deuteration distribution estimation was retained in Hexicon 2 and improved using an iterative deconvolution algorithm that is robust even to noisy data. In addition, Hexicon 2 provides a data browser that facilitates quality control and provides convenient access to common data visualization tasks. Analysis of a benchmark dataset demonstrates superior performance of Hexicon 2 compared with its predecessor in terms of deuteration centroid recovery and deuteration distribution estimation. Hexicon 2 greatly reduces data analysis time compared with manual analysis, whereas the increased number of peptides provides redundant coverage of the entire protein sequence. Hexicon 2 is a standalone application available free of charge under http://hx2.mpimf-heidelberg.mpg.de.
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Lindner, Robert; Lou, Xinghua; Reinstein, Jochen; Shoeman, Robert L; Hamprecht, Fred A; Winkler, Andreas
2014-06-01
Hydrogen-deuterium exchange (HDX) experiments analyzed by mass spectrometry (MS) provide information about the dynamics and the solvent accessibility of protein backbone amide hydrogen atoms. Continuous improvement of MS instrumentation has contributed to the increasing popularity of this method; however, comprehensive automated data analysis is only beginning to mature. We present Hexicon 2, an automated pipeline for data analysis and visualization based on the previously published program Hexicon (Lou et al. 2010). Hexicon 2 employs the sensitive NITPICK peak detection algorithm of its predecessor in a divide-and-conquer strategy and adds new features, such as chromatogram alignment and improved peptide sequence assignment. The unique feature of deuteration distribution estimation was retained in Hexicon 2 and improved using an iterative deconvolution algorithm that is robust even to noisy data. In addition, Hexicon 2 provides a data browser that facilitates quality control and provides convenient access to common data visualization tasks. Analysis of a benchmark dataset demonstrates superior performance of Hexicon 2 compared with its predecessor in terms of deuteration centroid recovery and deuteration distribution estimation. Hexicon 2 greatly reduces data analysis time compared with manual analysis, whereas the increased number of peptides provides redundant coverage of the entire protein sequence. Hexicon 2 is a standalone application available free of charge under http://hx2.mpimf-heidelberg.mpg.de.
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The History of Bordetella pertussis Genome Evolution Includes Structural Rearrangement
Peng, Yanhui; Loparev, Vladimir; Batra, Dhwani; Bowden, Katherine E.; Burroughs, Mark; Cassiday, Pamela K.; Davis, Jamie K.; Johnson, Taccara; Juieng, Phalasy; Knipe, Kristen; Mathis, Marsenia H.; Pruitt, Andrea M.; Rowe, Lori; Sheth, Mili; Tondella, M. Lucia; Williams, Margaret M.
2017-01-01
ABSTRACT Despite high pertussis vaccine coverage, reported cases of whooping cough (pertussis) have increased over the last decade in the United States and other developed countries. Although Bordetella pertussis is well known for its limited gene sequence variation, recent advances in long-read sequencing technology have begun to reveal genomic structural heterogeneity among otherwise indistinguishable isolates, even within geographically or temporally defined epidemics. We have compared rearrangements among complete genome assemblies from 257 B. pertussis isolates to examine the potential evolution of the chromosomal structure in a pathogen with minimal gene nucleotide sequence diversity. Discrete changes in gene order were identified that differentiated genomes from vaccine reference strains and clinical isolates of various genotypes, frequently along phylogenetic boundaries defined by single nucleotide polymorphisms. The observed rearrangements were primarily large inversions centered on the replication origin or terminus and flanked by IS481, a mobile genetic element with >240 copies per genome and previously suspected to mediate rearrangements and deletions by homologous recombination. These data illustrate that structural genome evolution in B. pertussis is not limited to reduction but also includes rearrangement. Therefore, although genomes of clinical isolates are structurally diverse, specific changes in gene order are conserved, perhaps due to positive selection, providing novel information for investigating disease resurgence and molecular epidemiology. IMPORTANCE Whooping cough, primarily caused by Bordetella pertussis, has resurged in the United States even though the coverage with pertussis-containing vaccines remains high. The rise in reported cases has included increased disease rates among all vaccinated age groups, provoking questions about the pathogen's evolution. The chromosome of B. pertussis includes a large number of repetitive mobile genetic elements that obstruct genome analysis. However, these mobile elements facilitate large rearrangements that alter the order and orientation of essential protein-encoding genes, which otherwise exhibit little nucleotide sequence diversity. By comparing the complete genome assemblies from 257 isolates, we show that specific rearrangements have been conserved throughout recent evolutionary history, perhaps by eliciting changes in gene expression, which may also provide useful information for molecular epidemiology. PMID:28167525
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Sequence Complexity of Amyloidogenic Regions in Intrinsically Disordered Human Proteins
Das, Swagata; Pal, Uttam; Das, Supriya; Bagga, Khyati; Roy, Anupam; Mrigwani, Arpita; Maiti, Nakul C.
2014-01-01
An amyloidogenic region (AR) in a protein sequence plays a significant role in protein aggregation and amyloid formation. We have investigated the sequence complexity of AR that is present in intrinsically disordered human proteins. More than 80% human proteins in the disordered protein databases (DisProt+IDEAL) contained one or more ARs. With decrease of protein disorder, AR content in the protein sequence was decreased. A probability density distribution analysis and discrete analysis of AR sequences showed that ∼8% residue in a protein sequence was in AR and the region was in average 8 residues long. The residues in the AR were high in sequence complexity and it seldom overlapped with low complexity regions (LCR), which was largely abundant in disorder proteins. The sequences in the AR showed mixed conformational adaptability towards α-helix, β-sheet/strand and coil conformations. PMID:24594841
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How Many Protein Sequences Fold to a Given Structure? A Coevolutionary Analysis.
Tian, Pengfei; Best, Robert B
2017-10-17
Quantifying the relationship between protein sequence and structure is key to understanding the protein universe. A fundamental measure of this relationship is the total number of amino acid sequences that can fold to a target protein structure, known as the "sequence capacity," which has been suggested as a proxy for how designable a given protein fold is. Although sequence capacity has been extensively studied using lattice models and theory, numerical estimates for real protein structures are currently lacking. In this work, we have quantitatively estimated the sequence capacity of 10 proteins with a variety of different structures using a statistical model based on residue-residue co-evolution to capture the variation of sequences from the same protein family. Remarkably, we find that even for the smallest protein folds, such as the WW domain, the number of foldable sequences is extremely large, exceeding the Avogadro constant. In agreement with earlier theoretical work, the calculated sequence capacity is positively correlated with the size of the protein, or better, the density of contacts. This allows the absolute sequence capacity of a given protein to be approximately predicted from its structure. On the other hand, the relative sequence capacity, i.e., normalized by the total number of possible sequences, is an extremely tiny number and is strongly anti-correlated with the protein length. Thus, although there may be more foldable sequences for larger proteins, it will be much harder to find them. Lastly, we have correlated the evolutionary age of proteins in the CATH database with their sequence capacity as predicted by our model. The results suggest a trade-off between the opposing requirements of high designability and the likelihood of a novel fold emerging by chance. Published by Elsevier Inc.
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An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions.
Ruan, Guihua; Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui; Du, Fuyou
2016-04-22
A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of Nα-benzoyl-l-arginine ethyl ester to Nα-benzoyl-l-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. Copyright © 2016 Elsevier Inc. All rights reserved.
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Coprolites as a source of information on the genome and diet of the cave hyena
Bon, Céline; Berthonaud, Véronique; Maksud, Frédéric; Labadie, Karine; Poulain, Julie; Artiguenave, François; Wincker, Patrick; Aury, Jean-Marc; Elalouf, Jean-Marc
2012-01-01
We performed high-throughput sequencing of DNA from fossilized faeces to evaluate this material as a source of information on the genome and diet of Pleistocene carnivores. We analysed coprolites derived from the extinct cave hyena (Crocuta crocuta spelaea), and sequenced 90 million DNA fragments from two specimens. The DNA reads enabled a reconstruction of the cave hyena mitochondrial genome with up to a 158-fold coverage. This genome, and those sequenced from extant spotted (Crocuta crocuta) and striped (Hyaena hyaena) hyena specimens, allows for the establishment of a robust phylogeny that supports a close relationship between the cave and the spotted hyena. We also demonstrate that high-throughput sequencing yields data for cave hyena multi-copy and single-copy nuclear genes, and that about 50 per cent of the coprolite DNA can be ascribed to this species. Analysing the data for additional species to indicate the cave hyena diet, we retrieved abundant sequences for the red deer (Cervus elaphus), and characterized its mitochondrial genome with up to a 3.8-fold coverage. In conclusion, we have demonstrated the presence of abundant ancient DNA in the coprolites surveyed. Shotgun sequencing of this material yielded a wealth of DNA sequences for a Pleistocene carnivore and allowed unbiased identification of diet. PMID:22456883
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Scaglione, Davide; Reyes-Chin-Wo, Sebastian; Acquadro, Alberto; Froenicke, Lutz; Portis, Ezio; Beitel, Christopher; Tirone, Matteo; Mauro, Rosario; Lo Monaco, Antonino; Mauromicale, Giovanni; Faccioli, Primetta; Cattivelli, Luigi; Rieseberg, Loren; Michelmore, Richard; Lanteri, Sergio
2016-01-01
Globe artichoke (Cynara cardunculus var. scolymus) is an out-crossing, perennial, multi-use crop species that is grown worldwide and belongs to the Compositae, one of the most successful Angiosperm families. We describe the first genome sequence of globe artichoke. The assembly, comprising of 13,588 scaffolds covering 725 of the 1,084 Mb genome, was generated using ~133-fold Illumina sequencing data and encodes 26,889 predicted genes. Re-sequencing (30×) of globe artichoke and cultivated cardoon (C. cardunculus var. altilis) parental genotypes and low-coverage (0.5 to 1×) genotyping-by-sequencing of 163 F1 individuals resulted in 73% of the assembled genome being anchored in 2,178 genetic bins ordered along 17 chromosomal pseudomolecules. This was achieved using a novel pipeline, SOILoCo (Scaffold Ordering by Imputation with Low Coverage), to detect heterozygous regions and assign parental haplotypes with low sequencing read depth and of unknown phase. SOILoCo provides a powerful tool for de novo genome analysis of outcrossing species. Our data will enable genome-scale analyses of evolutionary processes among crops, weeds, and wild species within and beyond the Compositae, and will facilitate the identification of economically important genes from related species. PMID:26786968
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Lucas Lledó, José Ignacio; Cáceres, Mario
2013-01-01
One of the most used techniques to study structural variation at a genome level is paired-end mapping (PEM). PEM has the advantage of being able to detect balanced events, such as inversions and translocations. However, inversions are still quite difficult to predict reliably, especially from high-throughput sequencing data. We simulated realistic PEM experiments with different combinations of read and library fragment lengths, including sequencing errors and meaningful base-qualities, to quantify and track down the origin of false positives and negatives along sequencing, mapping, and downstream analysis. We show that PEM is very appropriate to detect a wide range of inversions, even with low coverage data. However, % of inversions located between segmental duplications are expected to go undetected by the most common sequencing strategies. In general, longer DNA libraries improve the detectability of inversions far better than increments of the coverage depth or the read length. Finally, we review the performance of three algorithms to detect inversions —SVDetect, GRIAL, and VariationHunter—, identify common pitfalls, and reveal important differences in their breakpoint precisions. These results stress the importance of the sequencing strategy for the detection of structural variants, especially inversions, and offer guidelines for the design of future genome sequencing projects. PMID:23637806
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A Pan-HIV Strategy for Complete Genome Sequencing
Yamaguchi, Julie; Alessandri-Gradt, Elodie; Tell, Robert W.; Brennan, Catherine A.
2015-01-01
Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5′ end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance. PMID:26699702
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Dong, Jia-Jia; Guan, De-Long; Xu, Sheng-Quan
2016-09-01
The complete mitogenome of Oxya intricate (Stål.) has been reconstructed from whole-genome Illumina sequencing data with an average coverage of 294×. The circular genome is 15,466 bp in length, and consists of 22 transfer RNAs (tRNAs), 13 protein-coding genes (PCGs), 2 ribosomal RNAs (rRNAs) and 1 D-loop region. All PCGs are initiated with ATN codons, and are terminated with TAR codons except for ND5 with the incomplete stop codon T. The nucleotide composition is asymmetric (42.5%A, 14.6%C, 10.6%G, 32.3%T) with an overall GC content of 25.2%. These data would contribute to the design of novel molecular markers for population and evolutionary studies of this and related orthopteran species.
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Ying, Mengchao; Kidou, Shin-Ichiro
2017-07-01
To adapt to cold conditions, barley plants rely on specific mechanisms, which have not been fully understood. In this study, we characterized a novel barley cold-induced gene identified using a PCR-based high coverage gene expression profiling method. The identified gene encodes a small protein that we named CISP1 (Cold-induced Small Protein 1). Homology searches of sequence databases revealed that CISP1 homologs (CISP2 and CISP3) exist in barley genome. Further database analyses showed that the CISP1 homologs were widely distributed in cold-tolerant plants such as wheat and rye. Quantitative reverse transcription PCR analyses indicated that the expression of barley CISP genes was markedly increased in roots exposed to cold conditions. In situ hybridization analyses showed that the CISP1 transcripts were localized in the root tip and lateral root primordium. We also demonstrated that the CISP1 protein bound to RNA. Taken together, these findings indicate that CISP1 and its homologs encoding small RNA-binding proteins may serve as RNA chaperones playing a vital role in the cold adaptation of barley root. This is the first report describing the likely close relationship between root-specific genes and the cold adaptation process, as well as the potential function of the identified genes. Copyright © 2017 Elsevier B.V. All rights reserved.
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Identification of Bacillus thuringiensis Cry1AbMod binding-proteins from Spodoptera frugiperda.
Martínez de Castro, Diana L; García-Gómez, Blanca I; Gómez, Isabel; Bravo, Alejandra; Soberón, Mario
2017-12-01
Bacillus thuringiensis Cry toxins are currently used for pest control in transgenic crops but evolution of resistance by the insect pests threatens the use of this technology. The Cry1AbMod toxin was engineered to lack the alpha helix-1 of the parental Cry1Ab toxin and was shown to counter resistance to Cry1Ab and Cry1Ac toxins in different insect species including the fall armyworm Spodoptera frugiperda. In addition, Cry1AbMod showed enhanced toxicity to Cry1Ab-susceptible S. frugiperda populations. To gain insights into the mechanisms of this Cry1AbMod-enhanced toxicity, we isolated the Cry1AbMod toxin binding proteins from S. frugiperda brush border membrane vesicles (BBMV), which were identified by pull-down assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS results indicated that Cry1AbMod toxin could bind to four classes of aminopeptidase (N1, N3, N4 y N5) and actin, with the highest amino acid sequence coverage acquired for APN 1 and APN4. In addition to these proteins, we found other proteins not previously described as Cry toxin binding proteins. This is the first report that suggests the interaction between Cry1AbMod and APN in S. frugiperda. Copyright © 2017 Elsevier Inc. All rights reserved.
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Studying the effect of microwave heating on the digestion process and identification of proteins.
Devi, Shobha; Wu, Bo-Hung; Chu, Pei-Yu; Liu, Yue-Pei; Wu, Hsin-Lin; Ho, Yen-Peng
2017-02-01
The impact of microwave irradiation on the in-solution digestion processes and the detection limit of proteins are systematically studied. Kinetic processes of many peptides produced through the trypsin digestion of various proteins under microwave heating at 50°C were investigated with MALDI-MS. This study also examines the detection limits and digestion completeness of individual proteins under microwave heating at 50°C and at different time intervals (1, 5 and 30 min) using LC-MS. We conclude that if the peptides without missed cleavage dictate the detection limit, conventional digestion will lead to a better detection limit. The detection limit may not differ between the microwave and conventional heating if the peptides with missed cleavage sites and strong intensity are formed at the very early stage (i.e., less than 1 min) and are not further digested throughout the entire digestion process. The digestion of Escherichia coli lysate was compared under conventional and short time (microwave) conditions. The number of proteins identified under conventional heating exceeded that obtained from microwave heating over heating periods less than 5 min. The overall results show that the microwave-assisted digestion is not complete. Although the sequence coverage might be better, the detection limit might be worse than that under conventional heating. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Characterization of mango (Mangifera indica L.) transcriptome and chloroplast genome.
Azim, M Kamran; Khan, Ishtaiq A; Zhang, Yong
2014-05-01
We characterized mango leaf transcriptome and chloroplast genome using next generation DNA sequencing. The RNA-seq output of mango transcriptome generated >12 million reads (total nucleotides sequenced >1 Gb). De novo transcriptome assembly generated 30,509 unigenes with lengths in the range of 300 to ≥3,000 nt and 67× depth of coverage. Blast searching against nonredundant nucleotide databases and several Viridiplantae genomic datasets annotated 24,593 mango unigenes (80% of total) and identified Citrus sinensis as closest neighbor of mango with 9,141 (37%) matched sequences. The annotation with gene ontology and Clusters of Orthologous Group terms categorized unigene sequences into 57 and 25 classes, respectively. More than 13,500 unigenes were assigned to 293 KEGG pathways. Besides major plant biology related pathways, KEGG based gene annotation pointed out active presence of an array of biochemical pathways involved in (a) biosynthesis of bioactive flavonoids, flavones and flavonols, (b) biosynthesis of terpenoids and lignins and (c) plant hormone signal transduction. The mango transcriptome sequences revealed 235 proteases belonging to five catalytic classes of proteolytic enzymes. The draft genome of mango chloroplast (cp) was obtained by a combination of Sanger and next generation sequencing. The draft mango cp genome size is 151,173 bp with a pair of inverted repeats of 27,093 bp separated by small and large single copy regions, respectively. Out of 139 genes in mango cp genome, 91 found to be protein coding. Sequence analysis revealed cp genome of C. sinensis as closest neighbor of mango. We found 51 short repeats in mango cp genome supposed to be associated with extensive rearrangements. This is the first report of transcriptome and chloroplast genome analysis of any Anacardiaceae family member.
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Garcillán-Barcia, M. Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M.; de la Cruz, Fernando
2014-01-01
Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ–proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages. PMID:25522143
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Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando
2014-12-01
Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.
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Philipp, E E R; Kraemer, L; Mountfort, D; Schilhabel, M; Schreiber, S; Rosenstiel, P
2012-03-15
Next generation sequencing (NGS) technologies allow a rapid and cost-effective compilation of large RNA sequence datasets in model and non-model organisms. However, the storage and analysis of transcriptome information from different NGS platforms is still a significant bottleneck, leading to a delay in data dissemination and subsequent biological understanding. Especially database interfaces with transcriptome analysis modules going beyond mere read counts are missing. Here, we present the Transcriptome Analysis and Comparison Explorer (T-ACE), a tool designed for the organization and analysis of large sequence datasets, and especially suited for transcriptome projects of non-model organisms with little or no a priori sequence information. T-ACE offers a TCL-based interface, which accesses a PostgreSQL database via a php-script. Within T-ACE, information belonging to single sequences or contigs, such as annotation or read coverage, is linked to the respective sequence and immediately accessible. Sequences and assigned information can be searched via keyword- or BLAST-search. Additionally, T-ACE provides within and between transcriptome analysis modules on the level of expression, GO terms, KEGG pathways and protein domains. Results are visualized and can be easily exported for external analysis. We developed T-ACE for laboratory environments, which have only a limited amount of bioinformatics support, and for collaborative projects in which different partners work on the same dataset from different locations or platforms (Windows/Linux/MacOS). For laboratories with some experience in bioinformatics and programming, the low complexity of the database structure and open-source code provides a framework that can be customized according to the different needs of the user and transcriptome project.
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The Caulobacter crescentus phage phiCbK: genomics of a canonical phage
2012-01-01
Background The bacterium Caulobacter crescentus is a popular model for the study of cell cycle regulation and senescence. The large prolate siphophage phiCbK has been an important tool in C. crescentus biology, and has been studied in its own right as a model for viral morphogenesis. Although a system of some interest, to date little genomic information is available on phiCbK or its relatives. Results Five novel phiCbK-like C. crescentus bacteriophages, CcrMagneto, CcrSwift, CcrKarma, CcrRogue and CcrColossus, were isolated from the environment. The genomes of phage phiCbK and these five environmental phage isolates were obtained by 454 pyrosequencing. The phiCbK-like phage genomes range in size from 205 kb encoding 318 proteins (phiCbK) to 280 kb encoding 448 proteins (CcrColossus), and were found to contain nonpermuted terminal redundancies of 10 to 17 kb. A novel method of terminal ligation was developed to map genomic termini, which confirmed termini predicted by coverage analysis. This suggests that sequence coverage discontinuities may be useable as predictors of genomic termini in phage genomes. Genomic modules encoding virion morphogenesis, lysis and DNA replication proteins were identified. The phiCbK-like phages were also found to encode a number of intriguing proteins; all contain a clearly T7-like DNA polymerase, and five of the six encode a possible homolog of the C. crescentus cell cycle regulator GcrA, which may allow the phage to alter the host cell’s replicative state. The structural proteome of phage phiCbK was determined, identifying the portal, major and minor capsid proteins, the tail tape measure and possible tail fiber proteins. All six phage genomes are clearly related; phiCbK, CcrMagneto, CcrSwift, CcrKarma and CcrRogue form a group related at the DNA level, while CcrColossus is more diverged but retains significant similarity at the protein level. Conclusions Due to their lack of any apparent relationship to other described phages, this group is proposed as the founding cohort of a new phage type, the phiCbK-like phages. This work will serve as a foundation for future studies on morphogenesis, infection and phage-host interactions in C. crescentus. PMID:23050599
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Yang, Xu; Lazar, Iulia M
2009-03-27
The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented. MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide. In this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing approximately 1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, y ions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW)<50,000. Preliminary experiments have demonstrated that putative biomarkers, that are not detectable by conventional data dependent MS acquisition methods in complex un-fractionated samples, can be reliable identified with the information provided in this library. Based on the spectral count, the quality of a tandem mass spectrum and the m/z values for a parent peptide and its most abundant daughter ions, MRM conditions can be selected to enable the detection of target peptides and proteins.
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2009-01-01
Background The discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented. Methods MCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide. Results In this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing ~1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, y ions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW)<50,000. Conclusion Preliminary experiments have demonstrated that putative biomarkers, that are not detectable by conventional data dependent MS acquisition methods in complex un-fractionated samples, can be reliable identified with the information provided in this library. Based on the spectral count, the quality of a tandem mass spectrum and the m/z values for a parent peptide and its most abundant daughter ions, MRM conditions can be selected to enable the detection of target peptides and proteins. PMID:19327145
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Enhancing genome assemblies by integrating non-sequence based data
2011-01-01
Introduction Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. Methods The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Results Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. Conclusions We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses. PMID:21554765
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Enhancing genome assemblies by integrating non-sequence based data.
Heider, Thomas N; Lindsay, James; Wang, Chenwei; O'Neill, Rachel J; Pask, Andrew J
2011-05-28
Many genome projects were underway before the advent of high-throughput sequencing and have thus been supported by a wealth of genome information from other technologies. Such information frequently takes the form of linkage and physical maps, both of which can provide a substantial amount of data useful in de novo sequencing projects. Furthermore, the recent abundance of genome resources enables the use of conserved synteny maps identified in related species to further enhance genome assemblies. The tammar wallaby (Macropus eugenii) is a model marsupial mammal with a low coverage genome. However, we have access to extensive comparative maps containing over 14,000 markers constructed through the physical mapping of conserved loci, chromosome painting and comprehensive linkage maps. Using a custom Bioperl pipeline, information from the maps was aligned to assembled tammar wallaby contigs using BLAT. This data was used to construct pseudo paired-end libraries with intervals ranging from 5-10 MB. We then used Bambus (a program designed to scaffold eukaryotic genomes by ordering and orienting contigs through the use of paired-end data) to scaffold our libraries. To determine how map data compares to sequence based approaches to enhance assemblies, we repeated the experiment using a 0.5× coverage of unique reads from 4 KB and 8 KB Illumina paired-end libraries. Finally, we combined both the sequence and non-sequence-based data to determine how a combined approach could further enhance the quality of the low coverage de novo reconstruction of the tammar wallaby genome. Using the map data alone, we were able order 2.2% of the initial contigs into scaffolds, and increase the N50 scaffold size to 39 KB (36 KB in the original assembly). Using only the 0.5× paired-end sequence based data, 53% of the initial contigs were assigned to scaffolds. Combining both data sets resulted in a further 2% increase in the number of initial contigs integrated into a scaffold (55% total) but a 35% increase in N50 scaffold size over the use of sequence-based data alone. We provide a relatively simple pipeline utilizing existing bioinformatics tools to integrate map data into a genome assembly which is available at http://www.mcb.uconn.edu/fac.php?name=paska. While the map data only contributed minimally to assigning the initial contigs to scaffolds in the new assembly, it greatly increased the N50 size. This process added structure to our low coverage assembly, greatly increasing its utility in further analyses.
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Bashyal, Bishnu M.; Rawat, Kirti; Sharma, Sapna; Kulshreshtha, Deepika; Gopala Krishnan, S.; Singh, Ashok K.; Dubey, Himanshu; Solanke, Amolkumar U.; Sharma, T. R.; Aggarwal, Rashmi
2017-01-01
Fusarium fujikuroi causing bakanae disease has emerged as one of the major pathogen of rice across the world. The study aims to comparative genomic analysis of Fusarium fujikuroi isolates and identification of the secretary proteins of the fungus involved in rice pathogenesis. In the present study, F. fujikuroi isolate “F250” was sequenced with an assembly size of 42.47 Mb providing coverage of 96.89% on reference IMI58289 genome. A total of 13,603 protein-coding genes were predicted from genome assembly. The average gene density in the F. fujikuroi genome was 315.10 genes per Mb with an average gene length of 1.67 kb. Additionally, 134,374 single nucleotide polymorphisms (SNPs) are identified against IMI58289 isolate, with an average SNP density of 3.11 per kb of genome. Repetitive elements represent approximately 270,550 bp, which is 0.63% of the total genome. In total, 3,109 simple sequence repeats (SSRs), including 302 compound SSRs are identified in the 8,656 scaffolds. Comparative analysis of the isolates of F. fujikuroi revealed that they shared a total of 12,240 common clusters with F250 showing higher similarity with IMI58289. A total of 1,194 secretory proteins were identified in its genome among which there were 356 genes encoding carbohydrate active enzymes (CAZymes) capable for degradation of complex polysaccharides. Out of them glycoside hydrolase (GH) families were most prevalent (41%) followed by carbohydrate esterase (CE). Out of them CE8 (4 genes), PL1 (10 genes), PL3 (5 genes), and GH28 (8 genes) were prominent plant cell wall degrading enzymes families in F250 secretome. Besides this, 585 genes essential for the pathogen–host interactions were also identified. Selected genes were validated through quantitative real-time PCR analyses in resistant and susceptible genotypes of rice at different days of inoculation. The data offers a better understanding of F. fujikuroi genome and will help us enhance our knowledge on Fusarium fujikuroi–rice interactions. PMID:29230233