A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI.

PubMed

Shinozuka, Hiroshi; Cogan, Noel O I; Shinozuka, Maiko; Marshall, Alexis; Kay, Pippa; Lin, Yi-Han; Spangenberg, German C; Forster, John W

2015-04-11

Fragmentation at random nucleotide locations is an essential process for preparation of DNA libraries to be used on massively parallel short-read DNA sequencing platforms. Although instruments for physical shearing, such as the Covaris S2 focused-ultrasonicator system, and products for enzymatic shearing, such as the Nextera technology and NEBNext dsDNA Fragmentase kit, are commercially available, a simple and inexpensive method is desirable for high-throughput sequencing library preparation. MspJI is a recently characterised restriction enzyme which recognises the sequence motif CNNR (where R = G or A) when the first base is modified to 5-methylcytosine or 5-hydroxymethylcytosine. A semi-random enzymatic DNA amplicon fragmentation method was developed based on the unique cleavage properties of MspJI. In this method, random incorporation of 5-methyl-2'-deoxycytidine-5'-triphosphate is achieved through DNA amplification with DNA polymerase, followed by DNA digestion with MspJI. Due to the recognition sequence of the enzyme, DNA amplicons are fragmented in a relatively sequence-independent manner. The size range of the resulting fragments was capable of control through optimisation of 5-methyl-2'-deoxycytidine-5'-triphosphate concentration in the reaction mixture. A library suitable for sequencing using the Illumina MiSeq platform was prepared and processed using the proposed method. Alignment of generated short reads to a reference sequence demonstrated a relatively high level of random fragmentation. The proposed method may be performed with standard laboratory equipment. Although the uniformity of coverage was slightly inferior to the Covaris physical shearing procedure, due to efficiencies of cost and labour, the method may be more suitable than existing approaches for implementation in large-scale sequencing activities, such as bacterial artificial chromosome (BAC)-based genome sequence assembly, pan-genomic studies and locus-targeted genotyping-by-sequencing.

Non-biological synthetic spike-in controls and the AMPtk software pipeline improve mycobiome data

Treesearch

Jonathan M. Palmer; Michelle A. Jusino; Mark T. Banik; Daniel L. Lindner

2018-01-01

High-throughput amplicon sequencing (HTAS) of conserved DNA regions is a powerful technique to characterize microbial communities. Recently, spike-in mock communities have been used to measure accuracy of sequencing platforms and data analysis pipelines. To assess the ability of sequencing platforms and data processing pipelines using fungal internal transcribed spacer...

[Current applications of high-throughput DNA sequencing technology in antibody drug research].

PubMed

Yu, Xin; Liu, Qi-Gang; Wang, Ming-Rong

2012-03-01

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

Two-color, 30 second microwave-accelerated Metal-Enhanced Fluorescence DNA assays: a new Rapid Catch and Signal (RCS) technology.

PubMed

Dragan, Anatoliy I; Golberg, Karina; Elbaz, Amit; Marks, Robert; Zhang, Yongxia; Geddes, Chris D

2011-03-07

For analyses of DNA fragment sequences in solution we introduce a 2-color DNA assay, utilizing a combination of the Metal-Enhanced Fluorescence (MEF) effect and microwave-accelerated DNA hybridization. The assay is based on a new "Catch and Signal" technology, i.e. the simultaneous specific recognition of two target DNA sequences in one well by complementary anchor-ssDNAs, attached to silver island films (SiFs). It is shown that fluorescent labels (Alexa 488 and Alexa 594), covalently attached to ssDNA fragments, play the role of biosensor recognition probes, demonstrating strong response upon DNA hybridization, locating fluorophores in close proximity to silver NPs, which is ideal for MEF. Subsequently the emission dramatically increases, while the excited state lifetime decreases. It is also shown that 30s microwave irradiation of wells, containing DNA molecules, considerably (~1000-fold) speeds up the highly selective hybridization of DNA fragments at ambient temperature. The 2-color "Catch and Signal" DNA assay platform can radically expedite quantitative analysis of genome DNA sequences, creating a simple and fast bio-medical platform for nucleic acid analysis. Copyright © 2010 Elsevier B.V. All rights reserved.

Species classifier choice is a key consideration when analysing low-complexity food microbiome data.

PubMed

Walsh, Aaron M; Crispie, Fiona; O'Sullivan, Orla; Finnegan, Laura; Claesson, Marcus J; Cotter, Paul D

2018-03-20

The use of shotgun metagenomics to analyse low-complexity microbial communities in foods has the potential to be of considerable fundamental and applied value. However, there is currently no consensus with respect to choice of species classification tool, platform, or sequencing depth. Here, we benchmarked the performances of three high-throughput short-read sequencing platforms, the Illumina MiSeq, NextSeq 500, and Ion Proton, for shotgun metagenomics of food microbiota. Briefly, we sequenced six kefir DNA samples and a mock community DNA sample, the latter constructed by evenly mixing genomic DNA from 13 food-related bacterial species. A variety of bioinformatic tools were used to analyse the data generated, and the effects of sequencing depth on these analyses were tested by randomly subsampling reads. Compositional analysis results were consistent between the platforms at divergent sequencing depths. However, we observed pronounced differences in the predictions from species classification tools. Indeed, PERMANOVA indicated that there was no significant differences between the compositional results generated by the different sequencers (p = 0.693, R 2  = 0.011), but there was a significant difference between the results predicted by the species classifiers (p = 0.01, R 2  = 0.127). The relative abundances predicted by the classifiers, apart from MetaPhlAn2, were apparently biased by reference genome sizes. Additionally, we observed varying false-positive rates among the classifiers. MetaPhlAn2 had the lowest false-positive rate, whereas SLIMM had the greatest false-positive rate. Strain-level analysis results were also similar across platforms. Each platform correctly identified the strains present in the mock community, but accuracy was improved slightly with greater sequencing depth. Notably, PanPhlAn detected the dominant strains in each kefir sample above 500,000 reads per sample. Again, the outputs from functional profiling analysis using SUPER-FOCUS were generally accordant between the platforms at different sequencing depths. Finally, and expectedly, metagenome assembly completeness was significantly lower on the MiSeq than either on the NextSeq (p = 0.03) or the Proton (p = 0.011), and it improved with increased sequencing depth. Our results demonstrate a remarkable similarity in the results generated by the three sequencing platforms at different sequencing depths, and, in fact, the choice of bioinformatics methodology had a more evident impact on results than the choice of sequencer did.

Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample.

PubMed

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.

Comparison of the Predictive Accuracy of DNA Array-Based Multigene Classifiers across cDNA Arrays and Affymetrix GeneChips

PubMed Central

Stec, James; Wang, Jing; Coombes, Kevin; Ayers, Mark; Hoersch, Sebastian; Gold, David L.; Ross, Jeffrey S; Hess, Kenneth R.; Tirrell, Stephen; Linette, Gerald; Hortobagyi, Gabriel N.; Symmans, W. Fraser; Pusztai, Lajos

2005-01-01

We examined how well differentially expressed genes and multigene outcome classifiers retain their class-discriminating values when tested on data generated by different transcriptional profiling platforms. RNA from 33 stage I-III breast cancers was hybridized to both Affymetrix GeneChip and Millennium Pharmaceuticals cDNA arrays. Only 30% of all corresponding gene expression measurements on the two platforms had Pearson correlation coefficient r ≥ 0.7 when UniGene was used to match probes. There was substantial variation in correlation between different Affymetrix probe sets matched to the same cDNA probe. When cDNA and Affymetrix probes were matched by basic local alignment tool (BLAST) sequence identity, the correlation increased substantially. We identified 182 genes in the Affymetrix and 45 in the cDNA data (including 17 common genes) that accurately separated 91% of cases in supervised hierarchical clustering in each data set. Cross-platform testing of these informative genes resulted in lower clustering accuracy of 45 and 79%, respectively. Several sets of accurate five-gene classifiers were developed on each platform using linear discriminant analysis. The best 100 classifiers showed average misclassification error rate of 2% on the original data that rose to 19.5% when tested on data from the other platform. Random five-gene classifiers showed misclassification error rate of 33%. We conclude that multigene predictors optimized for one platform lose accuracy when applied to data from another platform due to missing genes and sequence differences in probes that result in differing measurements for the same gene. PMID:16049308

Annotation-based genome-wide SNP discovery in the large and complex Aegilops tauschii genome using next-generation sequencing without a reference genome sequence

PubMed Central

2011-01-01

Background Many plants have large and complex genomes with an abundance of repeated sequences. Many plants are also polyploid. Both of these attributes typify the genome architecture in the tribe Triticeae, whose members include economically important wheat, rye and barley. Large genome sizes, an abundance of repeated sequences, and polyploidy present challenges to genome-wide SNP discovery using next-generation sequencing (NGS) of total genomic DNA by making alignment and clustering of short reads generated by the NGS platforms difficult, particularly in the absence of a reference genome sequence. Results An annotation-based, genome-wide SNP discovery pipeline is reported using NGS data for large and complex genomes without a reference genome sequence. Roche 454 shotgun reads with low genome coverage of one genotype are annotated in order to distinguish single-copy sequences and repeat junctions from repetitive sequences and sequences shared by paralogous genes. Multiple genome equivalents of shotgun reads of another genotype generated with SOLiD or Solexa are then mapped to the annotated Roche 454 reads to identify putative SNPs. A pipeline program package, AGSNP, was developed and used for genome-wide SNP discovery in Aegilops tauschii-the diploid source of the wheat D genome, and with a genome size of 4.02 Gb, of which 90% is repetitive sequences. Genomic DNA of Ae. tauschii accession AL8/78 was sequenced with the Roche 454 NGS platform. Genomic DNA and cDNA of Ae. tauschii accession AS75 was sequenced primarily with SOLiD, although some Solexa and Roche 454 genomic sequences were also generated. A total of 195,631 putative SNPs were discovered in gene sequences, 155,580 putative SNPs were discovered in uncharacterized single-copy regions, and another 145,907 putative SNPs were discovered in repeat junctions. These SNPs were dispersed across the entire Ae. tauschii genome. To assess the false positive SNP discovery rate, DNA containing putative SNPs was amplified by PCR from AL8/78 and AS75 and resequenced with the ABI 3730 xl. In a sample of 302 randomly selected putative SNPs, 84.0% in gene regions, 88.0% in repeat junctions, and 81.3% in uncharacterized regions were validated. Conclusion An annotation-based genome-wide SNP discovery pipeline for NGS platforms was developed. The pipeline is suitable for SNP discovery in genomic libraries of complex genomes and does not require a reference genome sequence. The pipeline is applicable to all current NGS platforms, provided that at least one such platform generates relatively long reads. The pipeline package, AGSNP, and the discovered 497,118 Ae. tauschii SNPs can be accessed at (http://avena.pw.usda.gov/wheatD/agsnp.shtml). PMID:21266061

Quality control of next-generation sequencing library through an integrative digital microfluidic platform.

PubMed

Thaitrong, Numrin; Kim, Hanyoup; Renzi, Ronald F; Bartsch, Michael S; Meagher, Robert J; Patel, Kamlesh D

2012-12-01

We have developed an automated quality control (QC) platform for next-generation sequencing (NGS) library characterization by integrating a droplet-based digital microfluidic (DMF) system with a capillary-based reagent delivery unit and a quantitative CE module. Using an in-plane capillary-DMF interface, a prepared sample droplet was actuated into position between the ground electrode and the inlet of the separation capillary to complete the circuit for an electrokinetic injection. Using a DNA ladder as an internal standard, the CE module with a compact LIF detector was capable of detecting dsDNA in the range of 5-100 pg/μL, suitable for the amount of DNA required by the Illumina Genome Analyzer sequencing platform. This DMF-CE platform consumes tenfold less sample volume than the current Agilent BioAnalyzer QC technique, preserving precious sample while providing necessary sensitivity and accuracy for optimal sequencing performance. The ability of this microfluidic system to validate NGS library preparation was demonstrated by examining the effects of limited-cycle PCR amplification on the size distribution and the yield of Illumina-compatible libraries, demonstrating that as few as ten cycles of PCR bias the size distribution of the library toward undesirable larger fragments. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reconstructing evolutionary trees in parallel for massive sequences.

PubMed

Zou, Quan; Wan, Shixiang; Zeng, Xiangxiang; Ma, Zhanshan Sam

2017-12-14

Building the evolutionary trees for massive unaligned DNA sequences is challenging and crucial. However, reconstructing evolutionary tree for ultra-large sequences is hard. Massive multiple sequence alignment is also challenging and time/space consuming. Hadoop and Spark are developed recently, which bring spring light for the classical computational biology problems. In this paper, we tried to solve the multiple sequence alignment and evolutionary reconstruction in parallel. HPTree, which is developed in this paper, can deal with big DNA sequence files quickly. It works well on the >1GB files, and gets better performance than other evolutionary reconstruction tools. Users could use HPTree for reonstructing evolutioanry trees on the computer clusters or cloud platform (eg. Amazon Cloud). HPTree could help on population evolution research and metagenomics analysis. In this paper, we employ the Hadoop and Spark platform and design an evolutionary tree reconstruction software tool for unaligned massive DNA sequences. Clustering and multiple sequence alignment are done in parallel. Neighbour-joining model was employed for the evolutionary tree building. We opened our software together with source codes via http://lab.malab.cn/soft/HPtree/ .

A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS

PubMed Central

Jiao, Xiaoli; Zheng, Xin; Ma, Liang; Kutty, Geetha; Gogineni, Emile; Sun, Qiang; Sherman, Brad T.; Hu, Xiaojun; Jones, Kristine; Raley, Castle; Tran, Bao; Munroe, David J.; Stephens, Robert; Liang, Dun; Imamichi, Tomozumi; Kovacs, Joseph A.; Lempicki, Richard A.; Huang, Da Wei

2013-01-01

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a De Novo assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results. PMID:24179701

A Benchmark Study on Error Assessment and Quality Control of CCS Reads Derived from the PacBio RS.

PubMed

Jiao, Xiaoli; Zheng, Xin; Ma, Liang; Kutty, Geetha; Gogineni, Emile; Sun, Qiang; Sherman, Brad T; Hu, Xiaojun; Jones, Kristine; Raley, Castle; Tran, Bao; Munroe, David J; Stephens, Robert; Liang, Dun; Imamichi, Tomozumi; Kovacs, Joseph A; Lempicki, Richard A; Huang, Da Wei

2013-07-31

PacBio RS, a newly emerging third-generation DNA sequencing platform, is based on a real-time, single-molecule, nano-nitch sequencing technology that can generate very long reads (up to 20-kb) in contrast to the shorter reads produced by the first and second generation sequencing technologies. As a new platform, it is important to assess the sequencing error rate, as well as the quality control (QC) parameters associated with the PacBio sequence data. In this study, a mixture of 10 prior known, closely related DNA amplicons were sequenced using the PacBio RS sequencing platform. After aligning Circular Consensus Sequence (CCS) reads derived from the above sequencing experiment to the known reference sequences, we found that the median error rate was 2.5% without read QC, and improved to 1.3% with an SVM based multi-parameter QC method. In addition, a De Novo assembly was used as a downstream application to evaluate the effects of different QC approaches. This benchmark study indicates that even though CCS reads are post error-corrected it is still necessary to perform appropriate QC on CCS reads in order to produce successful downstream bioinformatics analytical results.

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

PubMed Central

Fuller, Carl W.; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P. Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T.; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J.; Kasianowicz, John J.; Davis, Randy; Roever, Stefan; Church, George M.; Ju, Jingyue

2016-01-01

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. PMID:27091962

Reproducibility of Illumina platform deep sequencing errors allows accurate determination of DNA barcodes in cells.

PubMed

Beltman, Joost B; Urbanus, Jos; Velds, Arno; van Rooij, Nienke; Rohr, Jan C; Naik, Shalin H; Schumacher, Ton N

2016-04-02

Next generation sequencing (NGS) of amplified DNA is a powerful tool to describe genetic heterogeneity within cell populations that can both be used to investigate the clonal structure of cell populations and to perform genetic lineage tracing. For applications in which both abundant and rare sequences are biologically relevant, the relatively high error rate of NGS techniques complicates data analysis, as it is difficult to distinguish rare true sequences from spurious sequences that are generated by PCR or sequencing errors. This issue, for instance, applies to cellular barcoding strategies that aim to follow the amount and type of offspring of single cells, by supplying these with unique heritable DNA tags. Here, we use genetic barcoding data from the Illumina HiSeq platform to show that straightforward read threshold-based filtering of data is typically insufficient to filter out spurious barcodes. Importantly, we demonstrate that specific sequencing errors occur at an approximately constant rate across different samples that are sequenced in parallel. We exploit this observation by developing a novel approach to filter out spurious sequences. Application of our new method demonstrates its value in the identification of true sequences amongst spurious sequences in biological data sets.

Inaugural Genomics Automation Congress and the coming deluge of sequencing data.

PubMed

Creighton, Chad J

2010-10-01

Presentations at Select Biosciences's first 'Genomics Automation Congress' (Boston, MA, USA) in 2010 focused on next-generation sequencing and the platforms and methodology around them. The meeting provided an overview of sequencing technologies, both new and emerging. Speakers shared their recent work on applying sequencing to profile cells for various levels of biomolecular complexity, including DNA sequences, DNA copy, DNA methylation, mRNA and microRNA. With sequencing time and costs continuing to drop dramatically, a virtual explosion of very large sequencing datasets is at hand, which will probably present challenges and opportunities for high-level data analysis and interpretation, as well as for information technology infrastructure.

Niche and neutral processes both shape community structure in parallelized, aerobic, single carbon-source enrichments

DOE Data Explorer

Flynn, Theodore M.; Koval, Jason C.; Greenwald, Stephanie M.; Owens, Sarah M.; Kemner, Kenneth M.; Antonopoulos, Dionysios A.

2017-01-01

We present DNA sequence data in FASTA-formatted files from aerobic environmental microcosms inoculated with a sole carbon source. DNA sequences are of 16S rRNA genes present in DNA extracted from each microcosm along with the environmental samples (soil, water) used to inoculate them. These samples were sequenced using the Illumina MiSeq platform at the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory. This data is compatible with standard microbiome analysis pipelines (e.g., QIIME, mothur, etc.).

Next-Generation Sequencing Platforms

NASA Astrophysics Data System (ADS)

Mardis, Elaine R.

2013-06-01

Automated DNA sequencing instruments embody an elegant interplay among chemistry, engineering, software, and molecular biology and have built upon Sanger's founding discovery of dideoxynucleotide sequencing to perform once-unfathomable tasks. Combined with innovative physical mapping approaches that helped to establish long-range relationships between cloned stretches of genomic DNA, fluorescent DNA sequencers produced reference genome sequences for model organisms and for the reference human genome. New types of sequencing instruments that permit amazing acceleration of data-collection rates for DNA sequencing have been developed. The ability to generate genome-scale data sets is now transforming the nature of biological inquiry. Here, I provide an historical perspective of the field, focusing on the fundamental developments that predated the advent of next-generation sequencing instruments and providing information about how these instruments work, their application to biological research, and the newest types of sequencers that can extract data from single DNA molecules.

Programmable DNA-Guided Artificial Restriction Enzymes.

PubMed

Enghiad, Behnam; Zhao, Huimin

2017-05-19

Restriction enzymes are essential tools for recombinant DNA technology that have revolutionized modern biological research. However, they have limited sequence specificity and availability. Here we report a Pyrococcus furiosus Argonaute (PfAgo) based platform for generating artificial restriction enzymes (AREs) capable of recognizing and cleaving DNA sequences at virtually any arbitrary site and generating defined sticky ends of varying length. Short DNA guides are used to direct PfAgo to target sites for cleavage at high temperatures (>87 °C) followed by reannealing of the cleaved single stranded DNAs. We used this platform to generate over 18 AREs for DNA fingerprinting and molecular cloning of PCR-amplified or genomic DNAs. These AREs work as efficiently as their naturally occurring counterparts, and some of them even do not have any naturally occurring counterparts, demonstrating easy programmability, generality, versatility, and high efficiency for this new technology.

Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics.

PubMed

Hirsch, B; Endris, V; Lassmann, S; Weichert, W; Pfarr, N; Schirmacher, P; Kovaleva, V; Werner, M; Bonzheim, I; Fend, F; Sperveslage, J; Kaulich, K; Zacher, A; Reifenberger, G; Köhrer, K; Stepanow, S; Lerke, S; Mayr, T; Aust, D E; Baretton, G; Weidner, S; Jung, A; Kirchner, T; Hansmann, M L; Burbat, L; von der Wall, E; Dietel, M; Hummel, M

2018-04-01

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.

Synchronous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment based on a zwitterionic copper (II) metal-organic framework.

PubMed

Qiu, Gui-Hua; Weng, Zi-Hua; Hu, Pei-Pei; Duan, Wen-Jun; Xie, Bao-Ping; Sun, Bin; Tang, Xiao-Yan; Chen, Jin-Xiang

2018-04-01

From a three-dimensional (3D) metal-organic framework (MOF) of {[Cu(Cmdcp)(phen)(H 2 O)] 2 ·9H 2 O} n (1, H 3 CmdcpBr = N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide, phen = phenanthroline), a sensitive and selective fluorescence sensor has been developed for the simultaneous detection of ebolavirus conserved RNA sequences and ebolavirus-encoded microRNA-like (miRNA-like) fragment. The results from molecular dynamics simulation confirmed that MOF 1 absorbs carboxyfluorescein (FAM)-tagged and 5(6)-carboxyrhodamine, triethylammonium salt (ROX)-tagged probe ss-DNA (probe DNA, P-DNA) by π … π stacking and hydrogen bonding, as well as additional electrostatic interactions to form a sensing platform of P-DNAs@1 with quenched FAM and ROX fluorescence. In the presence of targeted ebolavirus conserved RNA sequences or ebolavirus-encoded miRNA-like fragment, the fluorophore-labeled P-DNA hybridizes with the analyte to give a P-DNA@RNA duplex and released from MOF 1, triggering a fluorescence recovery. Simultaneous detection of two target RNAs has also been realized by single and synchronous fluorescence analysis. The formed sensing platform shows high sensitivity for ebolavirus conserved RNA sequences and ebolavirus-encoded miRNA-like fragment with detection limits at the picomolar level and high selectivity without cross-reaction between the two probes. MOF 1 thus shows the potential as an effective fluorescent sensing platform for the synchronous detection of two ebolavirus-related sequences, and offer improved diagnostic accuracy of Ebola virus disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and validation of an rDNA operon based primer walking strategy applicable to de novo bacterial genome finishing

PubMed Central

Eastman, Alexander W.; Yuan, Ze-Chun

2015-01-01

Advances in sequencing technology have drastically increased the depth and feasibility of bacterial genome sequencing. However, little information is available that details the specific techniques and procedures employed during genome sequencing despite the large numbers of published genomes. Shotgun approaches employed by second-generation sequencing platforms has necessitated the development of robust bioinformatics tools for in silico assembly, and complete assembly is limited by the presence of repetitive DNA sequences and multi-copy operons. Typically, re-sequencing with multiple platforms and laborious, targeted Sanger sequencing are employed to finish a draft bacterial genome. Here we describe a novel strategy based on the identification and targeted sequencing of repetitive rDNA operons to expedite bacterial genome assembly and finishing. Our strategy was validated by finishing the genome of Paenibacillus polymyxa strain CR1, a bacterium with potential in sustainable agriculture and bio-based processes. An analysis of the 38 contigs contained in the P. polymyxa strain CR1 draft genome revealed 12 repetitive rDNA operons with varied intragenic and flanking regions of variable length, unanimously located at contig boundaries and within contig gaps. These highly similar but not identical rDNA operons were experimentally verified and sequenced simultaneously with multiple, specially designed primer sets. This approach also identified and corrected significant sequence rearrangement generated during the initial in silico assembly of sequencing reads. Our approach reduces the required effort associated with blind primer walking for contig assembly, increasing both the speed and feasibility of genome finishing. Our study further reinforces the notion that repetitive DNA elements are major limiting factors for genome finishing. Moreover, we provided a step-by-step workflow for genome finishing, which may guide future bacterial genome finishing projects. PMID:25653642

TIA: algorithms for development of identity-linked SNP islands for analysis by massively parallel DNA sequencing.

PubMed

Farris, M Heath; Scott, Andrew R; Texter, Pamela A; Bartlett, Marta; Coleman, Patricia; Masters, David

2018-04-11

Single nucleotide polymorphisms (SNPs) located within the human genome have been shown to have utility as markers of identity in the differentiation of DNA from individual contributors. Massively parallel DNA sequencing (MPS) technologies and human genome SNP databases allow for the design of suites of identity-linked target regions, amenable to sequencing in a multiplexed and massively parallel manner. Therefore, tools are needed for leveraging the genotypic information found within SNP databases for the discovery of genomic targets that can be evaluated on MPS platforms. The SNP island target identification algorithm (TIA) was developed as a user-tunable system to leverage SNP information within databases. Using data within the 1000 Genomes Project SNP database, human genome regions were identified that contain globally ubiquitous identity-linked SNPs and that were responsive to targeted resequencing on MPS platforms. Algorithmic filters were used to exclude target regions that did not conform to user-tunable SNP island target characteristics. To validate the accuracy of TIA for discovering these identity-linked SNP islands within the human genome, SNP island target regions were amplified from 70 contributor genomic DNA samples using the polymerase chain reaction. Multiplexed amplicons were sequenced using the Illumina MiSeq platform, and the resulting sequences were analyzed for SNP variations. 166 putative identity-linked SNPs were targeted in the identified genomic regions. Of the 309 SNPs that provided discerning power across individual SNP profiles, 74 previously undefined SNPs were identified during evaluation of targets from individual genomes. Overall, DNA samples of 70 individuals were uniquely identified using a subset of the suite of identity-linked SNP islands. TIA offers a tunable genome search tool for the discovery of targeted genomic regions that are scalable in the population frequency and numbers of SNPs contained within the SNP island regions. It also allows the definition of sequence length and sequence variability of the target region as well as the less variable flanking regions for tailoring to MPS platforms. As shown in this study, TIA can be used to discover identity-linked SNP islands within the human genome, useful for differentiating individuals by targeted resequencing on MPS technologies.

Colorimetric biosensing of targeted gene sequence using dual nanoparticle platforms

PubMed Central

Thavanathan, Jeevan; Huang, Nay Ming; Thong, Kwai Lin

2015-01-01

We have developed a colorimetric biosensor using a dual platform of gold nanoparticles and graphene oxide sheets for the detection of Salmonella enterica. The presence of the invA gene in S. enterica causes a change in color of the biosensor from its original pinkish-red to a light purplish solution. This occurs through the aggregation of the primary gold nanoparticles–conjugated DNA probe onto the surface of the secondary graphene oxide–conjugated DNA probe through DNA hybridization with the targeted DNA sequence. Spectrophotometry analysis showed a shift in wavelength from 525 nm to 600 nm with 1 μM of DNA target. Specificity testing revealed that the biosensor was able to detect various serovars of the S. enterica while no color change was observed with the other bacterial species. Sensitivity testing revealed the limit of detection was at 1 nM of DNA target. This proves the effectiveness of the biosensor in the detection of S. enterica through DNA hybridization. PMID:25897217

Use of the Minion nanopore sequencer for rapid sequencing of avian influenza virus isolates

USDA-ARS?s Scientific Manuscript database

A relatively new sequencing technology, the MinION nanopore sequencer, provides a platform that is smaller, faster, and cheaper than existing Next Generation Sequence (NGS) technologies. The MinION sequences of individual strands of DNA and can produce millions of sequencing reads. The cost of the s...

Alternative SNP detection platforms, HRM and biosensors, for varietal identification in Vitis vinifera L. using F3H and LDOX genes.

PubMed

Gomes, Sónia; Castro, Cláudia; Barrias, Sara; Pereira, Leonor; Jorge, Pedro; Fernandes, José R; Martins-Lopes, Paula

2018-04-11

The wine sector requires quick and reliable methods for Vitis vinifera L. varietal identification. The number of V. vinifera varieties is estimated in about 5,000 worldwide. Single Nucleotide Polymorphisms (SNPs) represent the most basic and abundant form of genetic sequence variation, being adequate for varietal discrimination. The aim of this work was to develop DNA-based assays suitable to detect SNP variation in V. vinifera, allowing varietal discrimination. Genotyping by sequencing allowed the detection of eleven SNPs on two genes of the anthocyanin pathway, the flavanone 3-hydroxylase (F3H, EC: 1.14.11.9), and the leucoanthocyanidin dioxygenase (LDOX, EC 1.14.11.19; synonym anthocyanidin synthase, ANS) in twenty V. vinifera varieties. Three High Resolution Melting (HRM) assays were designed based on the sequencing information, discriminating five of the 20 varieties: Alicante Bouschet, Donzelinho Tinto, Merlot, Moscatel Galego and Tinta Roriz. Sanger sequencing of the HRM assay products confirmed the HRM profiles. Three probes, with different lengths and sequences, were used as bio-recognition elements in an optical biosensor platform based on a long period grating (LPG) fiber optic sensor. The label free platform detected a difference of a single SNP using genomic DNA samples. The two different platforms were successfully applied for grapevine varietal identification.

Single molecule sequencing of the M13 virus genome without amplification

PubMed Central

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X.; Yan, Qin; Deem, Michael W.; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias. PMID:29253901

Single molecule sequencing of the M13 virus genome without amplification.

PubMed

Zhao, Luyang; Deng, Liwei; Li, Gailing; Jin, Huan; Cai, Jinsen; Shang, Huan; Li, Yan; Wu, Haomin; Xu, Weibin; Zeng, Lidong; Zhang, Renli; Zhao, Huan; Wu, Ping; Zhou, Zhiliang; Zheng, Jiao; Ezanno, Pierre; Yang, Andrew X; Yan, Qin; Deem, Michael W; He, Jiankui

2017-01-01

Next generation sequencing (NGS) has revolutionized life sciences research. However, GC bias and costly, time-intensive library preparation make NGS an ill fit for increasing sequencing demands in the clinic. A new class of third-generation sequencing platforms has arrived to meet this need, capable of directly measuring DNA and RNA sequences at the single-molecule level without amplification. Here, we use the new GenoCare single-molecule sequencing platform from Direct Genomics to sequence the genome of the M13 virus. Our platform detects single-molecule fluorescence by total internal reflection microscopy, with sequencing-by-synthesis chemistry. We sequenced the genome of M13 to a depth of 316x, with 100% coverage. We determined a consensus sequence accuracy of 100%. In contrast to GC bias inherent to NGS results, we demonstrated that our single-molecule sequencing method yields minimal GC bias.

Sequence analysis of the canine mitochondrial DNA control region from shed hair samples in criminal investigations.

PubMed

Berger, C; Berger, B; Parson, W

2012-01-01

In recent years, evidence from domestic dogs has increasingly been analyzed by forensic DNA testing. Especially, canine hairs have proved most suitable and practical due to the high rate of hair transfer occurring between dogs and humans. Starting with the description of a contamination-free sample handling procedure, we give a detailed workflow for sequencing hypervariable segments (HVS) of the mtDNA control region from canine evidence. After the hair material is lysed and the DNA extracted by Phenol/Chloroform, the amplification and sequencing strategy comprises the HVS I and II of the canine control region and is optimized for DNA of medium-to-low quality and quantity. The sequencing procedure is based on the Sanger Big-dye deoxy-terminator method and the separation of the sequencing reaction products is performed on a conventional multicolor fluorescence detection capillary electrophoresis platform. Finally, software-aided base calling and sequence interpretation are addressed exemplarily.

SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data

USGS Publications Warehouse

Miller, Mark P.; Knaus, Brian J.; Mullins, Thomas D.; Haig, Susan M.

2013-01-01

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

SSR_pipeline: a bioinformatic infrastructure for identifying microsatellites from paired-end Illumina high-throughput DNA sequencing data.

PubMed

Miller, Mark P; Knaus, Brian J; Mullins, Thomas D; Haig, Susan M

2013-01-01

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (e.g., microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains 3 analysis modules along with a fourth control module that can automate analyses of large volumes of data. The modules are used to 1) identify the subset of paired-end sequences that pass Illumina quality standards, 2) align paired-end reads into a single composite DNA sequence, and 3) identify sequences that possess microsatellites (both simple and compound) conforming to user-specified parameters. The microsatellite search algorithm is extremely efficient, and we have used it to identify repeats with motifs from 2 to 25 bp in length. Each of the 3 analysis modules can also be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc.). We demonstrate use of the program with data from the brine fly Ephydra packardi (Diptera: Ephydridae) and provide empirical timing benchmarks to illustrate program performance on a common desktop computer environment. We further show that the Illumina platform is capable of identifying large numbers of microsatellites, even when using unenriched sample libraries and a very small percentage of the sequencing capacity from a single DNA sequencing run. All modules from SSR_pipeline are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, and Windows).

Towards Clinical Molecular Diagnosis of Inherited Cardiac Conditions: A Comparison of Bench-Top Genome DNA Sequencers

PubMed Central

Wilkinson, Samuel L.; John, Shibu; Walsh, Roddy; Novotny, Tomas; Valaskova, Iveta; Gupta, Manu; Game, Laurence; Barton, Paul J R.; Cook, Stuart A.; Ware, James S.

2013-01-01

Background Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations. Methodology/Principal Findings We evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS. Conclusions/Significance MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive. PMID:23861798

Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

PubMed

Cartwright, Joseph F; Anderson, Karin; Longworth, Joseph; Lobb, Philip; James, David C

2018-06-01

High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process. © 2018 Wiley Periodicals, Inc.

Multiple advanced logic gates made of DNA-Ag nanocluster and the application for intelligent detection of pathogenic bacterial genes† †Electronic supplementary information (ESI) available: Chemicals, materials and DNA sequences used in the investigation, the construction of YES, AND, OR, XOR and INH logic gates, CD and PAGE experimental results. See DOI: 10.1039/c7sc05246d

PubMed Central

Lin, Xiaodong; Deng, Jiankang; Lyu, Yanlong; Qian, Pengcheng; Li, Yunfei

2018-01-01

The integration of multiple DNA logic gates on a universal platform to implement advance logic functions is a critical challenge for DNA computing. Herein, a straightforward and powerful strategy in which a guanine-rich DNA sequence lighting up a silver nanocluster and fluorophore was developed to construct a library of logic gates on a simple DNA-templated silver nanoclusters (DNA-AgNCs) platform. This library included basic logic gates, YES, AND, OR, INHIBIT, and XOR, which were further integrated into complex logic circuits to implement diverse advanced arithmetic/non-arithmetic functions including half-adder, half-subtractor, multiplexer, and demultiplexer. Under UV irradiation, all the logic functions could be instantly visualized, confirming an excellent repeatability. The logic operations were entirely based on DNA hybridization in an enzyme-free and label-free condition, avoiding waste accumulation and reducing cost consumption. Interestingly, a DNA-AgNCs-based multiplexer was, for the first time, used as an intelligent biosensor to identify pathogenic genes, E. coli and S. aureus genes, with a high sensitivity. The investigation provides a prototype for the wireless integration of multiple devices on even the simplest single-strand DNA platform to perform diverse complex functions in a straightforward and cost-effective way. PMID:29675221

Abnormal plasma DNA profiles in early ovarian cancer using a non-invasive prenatal testing platform: implications for cancer screening.

PubMed

Cohen, Paul A; Flowers, Nicola; Tong, Stephen; Hannan, Natalie; Pertile, Mark D; Hui, Lisa

2016-08-24

Non-invasive prenatal testing (NIPT) identifies fetal aneuploidy by sequencing cell-free DNA in the maternal plasma. Pre-symptomatic maternal malignancies have been incidentally detected during NIPT based on abnormal genomic profiles. This low coverage sequencing approach could have potential for ovarian cancer screening in the non-pregnant population. Our objective was to investigate whether plasma DNA sequencing with a clinical whole genome NIPT platform can detect early- and late-stage high-grade serous ovarian carcinomas (HGSOC). This is a case control study of prospectively-collected biobank samples comprising preoperative plasma from 32 women with HGSOC (16 'early cancer' (FIGO I-II) and 16 'advanced cancer' (FIGO III-IV)) and 32 benign controls. Plasma DNA from cases and controls were sequenced using a commercial NIPT platform and chromosome dosage measured. Sequencing data were blindly analyzed with two methods: (1) Subchromosomal changes were called using an open source algorithm WISECONDOR (WIthin-SamplE COpy Number aberration DetectOR). Genomic gains or losses ≥ 15 Mb were prespecified as "screen positive" calls, and mapped to recurrent copy number variations reported in an ovarian cancer genome atlas. (2) Selected whole chromosome gains or losses were reported using the routine NIPT pipeline for fetal aneuploidy. We detected 13/32 cancer cases using the subchromosomal analysis (sensitivity 40.6 %, 95 % CI, 23.7-59.4 %), including 6/16 early and 7/16 advanced HGSOC cases. Two of 32 benign controls had subchromosomal gains ≥ 15 Mb (specificity 93.8 %, 95 % CI, 79.2-99.2 %). Twelve of the 13 true positive cancer cases exhibited specific recurrent changes reported in HGSOC tumors. The NIPT pipeline resulted in one "monosomy 18" call from the cancer group, and two "monosomy X" calls in the controls. Low coverage plasma DNA sequencing used for prenatal testing detected 40.6 % of all HGSOC, including 38 % of early stage cases. Our findings demonstrate the potential of a high throughput sequencing platform to screen for early HGSOC in plasma based on characteristic multiple segmental chromosome gains and losses. The performance of this approach may be further improved by refining bioinformatics algorithms and targeting selected cancer copy number variations.

Integrating DNA strand displacement circuitry to the nonlinear hybridization chain reaction.

PubMed

Zhang, Zhuo; Fan, Tsz Wing; Hsing, I-Ming

2017-02-23

Programmable and modular attributes of DNA molecules allow one to develop versatile sensing platforms that can be operated isothermally and enzyme-free. In this work, we present an approach to integrate upstream DNA strand displacement circuits that can be turned on by a sequence-specific microRNA analyte with a downstream nonlinear hybridization chain reaction for a cascading hyperbranched nucleic acid assembly. This system provides a two-step amplification strategy for highly sensitive detection of the miRNA analyte, conducive for multiplexed detection. Multiple miRNA analytes were tested with our integrated circuitry using the same downstream signal amplification setting, showing the decoupling of nonlinear self-assembly with the analyte sequence. Compared with the reported methods, our signal amplification approach provides an additional control module for higher-order DNA self-assembly and could be developed into a promising platform for the detection of critical nucleic-acid based biomarkers.

Salient Features of Endonuclease Platforms for Therapeutic Genome Editing.

PubMed

Certo, Michael T; Morgan, Richard A

2016-03-01

Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications.

Salient Features of Endonuclease Platforms for Therapeutic Genome Editing

PubMed Central

Certo, Michael T; Morgan, Richard A

2016-01-01

Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications. PMID:26796671

Genetic Constructor: An Online DNA Design Platform.

PubMed

Bates, Maxwell; Lachoff, Joe; Meech, Duncan; Zulkower, Valentin; Moisy, Anaïs; Luo, Yisha; Tekotte, Hille; Franziska Scheitz, Cornelia Johanna; Khilari, Rupal; Mazzoldi, Florencio; Chandran, Deepak; Groban, Eli

2017-12-15

Genetic Constructor is a cloud Computer Aided Design (CAD) application developed to support synthetic biologists from design intent through DNA fabrication and experiment iteration. The platform allows users to design, manage, and navigate complex DNA constructs and libraries, using a new visual language that focuses on functional parts abstracted from sequence. Features like combinatorial libraries and automated primer design allow the user to separate design from construction by focusing on functional intent, and design constraints aid iterative refinement of designs. A plugin architecture enables contributions from scientists and coders to leverage existing powerful software and connect to DNA foundries. The software is easily accessible and platform agnostic, free for academics, and available in an open-source community edition. Genetic Constructor seeks to democratize DNA design, manufacture, and access to tools and services from the synthetic biology community.

Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs).

PubMed

Cantsilieris, Stuart; Stessman, Holly A; Shendure, Jay; Eichler, Evan E

2017-01-01

Molecular inversion probes (MIPs) in combination with massively parallel DNA sequencing represent a versatile, yet economical tool for targeted sequencing of genomic DNA. Several thousand genomic targets can be selectively captured using long oligonucleotides containing unique targeting arms and universal linkers. The ability to append sequencing adaptors and sample-specific barcodes allows large-scale pooling and subsequent high-throughput sequencing at relatively low cost per sample. Here, we describe a "wet bench" protocol detailing the capture and subsequent sequencing of >2000 genomic targets from 192 samples, representative of a single lane on the Illumina HiSeq 2000 platform.

Fabrication and characterization of a solid state nanopore with self-aligned carbon nanoelectrodes for molecular detection

NASA Astrophysics Data System (ADS)

Spinney, Patrick; Collins, Scott D.; Howitt, David G.; Smith, Rosemary L.

2012-06-01

Rapid and cost-effective DNA sequencing is a pivotal prerequisite for the genomics era. Many of the recent advances in forensics, medicine, agriculture, taxonomy, and drug discovery have paralleled critical advances in DNA sequencing technology. Nanopore modalities for DNA sequencing have recently surfaced including the electrical interrogation of protein ion channels and/or solid-state nanopores during translocation of DNA. However to date, most of this work has met with mixed success. In this work, we present a unique nanofabrication strategy that realizes an artificial nanopore articulated with carbon electrodes to sense the current modulations during the transport of DNA through the nanopore. This embodiment overcomes most of the technical difficulties inherent in other artificial nanopore embodiments and present a versatile platform for the testing of DNA single nucleotide detection. Characterization of the device using gold nanoparticles, silica nanoparticles, lambda dsDNA and 16-mer ssDNA are presented. Although single molecule DNA sequencing is still not demonstrated, the device shows a path towards this goal.

A graphene-based biosensing platform based on the release of DNA probes and rolling circle amplification.

PubMed

Liu, Meng; Song, Jinping; Shuang, Shaomin; Dong, Chuan; Brennan, John D; Li, Yingfu

2014-06-24

We report a versatile biosensing platform capable of achieving ultrasensitive detection of both small-molecule and macromolecular targets. The system features three components: reduced graphene oxide for its ability to adsorb single-stranded DNA molecules nonspecifically, DNA aptamers for their ability to bind reduced graphene oxide but undergo target-induced conformational changes that facilitate their release from the reduced graphene oxide surface, and rolling circle amplification (RCA) for its ability to amplify a primer-template recognition event into repetitive sequence units that can be easily detected. The key to the design is the tagging of a short primer to an aptamer sequence, which results in a small DNA probe that allows for both effective probe adsorption onto the reduced graphene oxide surface to mask the primer domain in the absence of the target, as well as efficient probe release in the presence of the target to make the primer available for template binding and RCA. We also made an observation that the circular template, which on its own does not cause a detectable level of probe release from the reduced graphene oxide, augments target-induced probe release. The synergistic release of DNA probes is interpreted to be a contributing factor for the high detection sensitivity. The broad utility of the platform is illustrated though engineering three different sensors that are capable of achieving ultrasensitive detection of a protein target, a DNA sequence and a small-molecule analyte. We envision that the approach described herein will find useful applications in the biological, medical, and environmental fields.

Picoliter DNA Sequencing Chemistry on an Electrowetting-based Digital Microfluidic Platform

PubMed Central

Ferguson Welch, Erin R.; Lin, Yan-You; Madison, Andrew; Fair, R.B.

2011-01-01

The results of investigations into performing DNA sequencing chemistry on a picoliter-scale electrowetting digital microfluidic platform are reported. Pyrosequencing utilizes pyrophosphate produced during nucleotide base addition to initiate a process ending with detection through a chemiluminescence reaction using firefly luciferase. The intensity of light produced during the reaction can be quantified to determine the number of bases added to the DNA strand. The logic-based control and discrete fluid droplets of a digital microfluidic device lend themselves well to the pyrosequencing process. Bead-bound DNA is magnetically held in a single location, and wash or reagent droplets added or split from it to circumvent product dilution. Here we discuss the dispensing, control, and magnetic manipulation of the paramagnetic beads used to hold target DNA. We also demonstrate and characterize the picoliter-scale reaction of luciferase with adenosine triphosphate to represent the detection steps of pyrosequencing and all necessary alterations for working on this scale. PMID:21298802

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling.

PubMed

Su, Jiao; Zhang, Haijie; Jiang, Bingying; Zheng, Huzhi; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

2011-11-15

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences. Copyright © 2011 Elsevier B.V. All rights reserved.

Genetic mutation analysis of human gastric adenocarcinomas using ion torrent sequencing platform.

PubMed

Xu, Zhi; Huo, Xinying; Ye, Hua; Tang, Chuanning; Nandakumar, Vijayalakshmi; Lou, Feng; Zhang, Dandan; Dong, Haichao; Sun, Hong; Jiang, Shouwen; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; He, Yan; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Gu, Dongying; Zhang, Xiaojing; Wu, Xiaomin; Wei, Xiaowei; Hong, Lingzhi; Zhang, Yangmei; Yang, Jinsong; Gong, Yonglin; Tang, Cuiju; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Chen, Jinfei

2014-01-01

Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.

An optimized and low-cost FPGA-based DNA sequence alignment--a step towards personal genomics.

PubMed

Shah, Hurmat Ali; Hasan, Laiq; Ahmad, Nasir

2013-01-01

DNA sequence alignment is a cardinal process in computational biology but also is much expensive computationally when performing through traditional computational platforms like CPU. Of many off the shelf platforms explored for speeding up the computation process, FPGA stands as the best candidate due to its performance per dollar spent and performance per watt. These two advantages make FPGA as the most appropriate choice for realizing the aim of personal genomics. The previous implementation of DNA sequence alignment did not take into consideration the price of the device on which optimization was performed. This paper presents optimization over previous FPGA implementation that increases the overall speed-up achieved as well as the price incurred by the platform that was optimized. The optimizations are (1) The array of processing elements is made to run on change in input value and not on clock, so eliminating the need for tight clock synchronization, (2) the implementation is unrestrained by the size of the sequences to be aligned, (3) the waiting time required for the sequences to load to FPGA is reduced to the minimum possible and (4) an efficient method is devised to store the output matrix that make possible to save the diagonal elements to be used in next pass, in parallel with the computation of output matrix. Implemented on Spartan3 FPGA, this implementation achieved 20 times performance improvement in terms of CUPS over GPP implementation.

Sequence Data for Clostridium autoethanogenum using Three Generations of Sequencing Technologies

DOE PAGES

Utturkar, Sagar M.; Klingeman, Dawn Marie; Bruno-Barcena, José M.; ...

2015-04-14

During the past decade, DNA sequencing output has been mostly dominated by the second generation sequencing platforms which are characterized by low cost, high throughput and shorter read lengths for example, Illumina. The emergence and development of so called third generation sequencing platforms such as PacBio has permitted exceptionally long reads (over 20 kb) to be generated. Due to read length increases, algorithm improvements and hybrid assembly approaches, the concept of one chromosome, one contig and automated finishing of microbial genomes is now a realistic and achievable task for many microbial laboratories. In this paper, we describe high quality sequencemore » datasets which span three generations of sequencing technologies, containing six types of data from four NGS platforms and originating from a single microorganism, Clostridium autoethanogenum. The dataset reported here will be useful for the scientific community to evaluate upcoming NGS platforms, enabling comparison of existing and novel bioinformatics approaches and will encourage interest in the development of innovative experimental and computational methods for NGS data.« less

A disruptive sequencer meets disruptive publishing.

PubMed

Loman, Nick; Goodwin, Sarah; Jansen, Hans; Loose, Matt

2015-01-01

Nanopore sequencing was recently made available to users in the form of the Oxford Nanopore MinION. Released to users through an early access programme, the MinION is made unique by its tiny form factor and ability to generate very long sequences from single DNA molecules. The platform is undergoing rapid evolution with three distinct nanopore types and five updates to library preparation chemistry in the last 18 months. To keep pace with the rapid evolution of this sequencing platform, and to provide a space where new analysis methods can be openly discussed, we present a new F1000Research channel devoted to updates to and analysis of nanopore sequence data.

TEA: the epigenome platform for Arabidopsis methylome study.

PubMed

Su, Sheng-Yao; Chen, Shu-Hwa; Lu, I-Hsuan; Chiang, Yih-Shien; Wang, Yu-Bin; Chen, Pao-Yang; Lin, Chung-Yen

2016-12-22

Bisulfite sequencing (BS-seq) has become a standard technology to profile genome-wide DNA methylation at single-base resolution. It allows researchers to conduct genome-wise cytosine methylation analyses on issues about genomic imprinting, transcriptional regulation, cellular development and differentiation. One single data from a BS-Seq experiment is resolved into many features according to the sequence contexts, making methylome data analysis and data visualization a complex task. We developed a streamlined platform, TEA, for analyzing and visualizing data from whole-genome BS-Seq (WGBS) experiments conducted in the model plant Arabidopsis thaliana. To capture the essence of the genome methylation level and to meet the efficiency for running online, we introduce a straightforward method for measuring genome methylation in each sequence context by gene. The method is scripted in Java to process BS-Seq mapping results. Through a simple data uploading process, the TEA server deploys a web-based platform for deep analysis by linking data to an updated Arabidopsis annotation database and toolkits. TEA is an intuitive and efficient online platform for analyzing the Arabidopsis genomic DNA methylation landscape. It provides several ways to help users exploit WGBS data. TEA is freely accessible for academic users at: http://tea.iis.sinica.edu.tw .

Ancient genomics

PubMed Central

Der Sarkissian, Clio; Allentoft, Morten E.; Ávila-Arcos, María C.; Barnett, Ross; Campos, Paula F.; Cappellini, Enrico; Ermini, Luca; Fernández, Ruth; da Fonseca, Rute; Ginolhac, Aurélien; Hansen, Anders J.; Jónsson, Hákon; Korneliussen, Thorfinn; Margaryan, Ashot; Martin, Michael D.; Moreno-Mayar, J. Víctor; Raghavan, Maanasa; Rasmussen, Morten; Velasco, Marcela Sandoval; Schroeder, Hannes; Schubert, Mikkel; Seguin-Orlando, Andaine; Wales, Nathan; Gilbert, M. Thomas P.; Willerslev, Eske; Orlando, Ludovic

2015-01-01

The past decade has witnessed a revolution in ancient DNA (aDNA) research. Although the field's focus was previously limited to mitochondrial DNA and a few nuclear markers, whole genome sequences from the deep past can now be retrieved. This breakthrough is tightly connected to the massive sequence throughput of next generation sequencing platforms and the ability to target short and degraded DNA molecules. Many ancient specimens previously unsuitable for DNA analyses because of extensive degradation can now successfully be used as source materials. Additionally, the analytical power obtained by increasing the number of sequence reads to billions effectively means that contamination issues that have haunted aDNA research for decades, particularly in human studies, can now be efficiently and confidently quantified. At present, whole genomes have been sequenced from ancient anatomically modern humans, archaic hominins, ancient pathogens and megafaunal species. Those have revealed important functional and phenotypic information, as well as unexpected adaptation, migration and admixture patterns. As such, the field of aDNA has entered the new era of genomics and has provided valuable information when testing specific hypotheses related to the past. PMID:25487338

Artificial Intelligence, DNA Mimicry, and Human Health.

PubMed

Stefano, George B; Kream, Richard M

2017-08-14

The molecular evolution of genomic DNA across diverse plant and animal phyla involved dynamic registrations of sequence modifications to maintain existential homeostasis to increasingly complex patterns of environmental stressors. As an essential corollary, driver effects of positive evolutionary pressure are hypothesized to effect concerted modifications of genomic DNA sequences to meet expanded platforms of regulatory controls for successful implementation of advanced physiological requirements. It is also clearly apparent that preservation of updated registries of advantageous modifications of genomic DNA sequences requires coordinate expansion of convergent cellular proofreading/error correction mechanisms that are encoded by reciprocally modified genomic DNA. Computational expansion of operationally defined DNA memory extends to coordinate modification of coding and previously under-emphasized noncoding regions that now appear to represent essential reservoirs of untapped genetic information amenable to evolutionary driven recruitment into the realm of biologically active domains. Additionally, expansion of DNA memory potential via chemical modification and activation of noncoding sequences is targeted to vertical augmentation and integration of an expanded cadre of transcriptional and epigenetic regulatory factors affecting linear coding of protein amino acid sequences within open reading frames.

Theory and modeling of particles with DNA-mediated interactions

NASA Astrophysics Data System (ADS)

Licata, Nicholas A.

2008-05-01

In recent years significant attention has been attracted to proposals which utilize DNA for nanotechnological applications. Potential applications of these ideas range from the programmable self-assembly of colloidal crystals, to biosensors and nanoparticle based drug delivery platforms. In Chapter I we introduce the system, which generically consists of colloidal particles functionalized with specially designed DNA markers. The sequence of bases on the DNA markers determines the particle type. Due to the hybridization between complementary single-stranded DNA, specific, type-dependent interactions can be introduced between particles by choosing the appropriate DNA marker sequences. In Chapter II we develop a statistical mechanical description of the aggregation and melting behavior of particles with DNA-mediated interactions. In Chapter III a model is proposed to describe the dynamical departure and diffusion of particles which form reversible key-lock connections. In Chapter IV we propose a method to self-assemble nanoparticle clusters using DNA scaffolds. A natural extension is discussed in Chapter V, the programmable self-assembly of nanoparticle clusters where the desired cluster geometry is encoded using DNA-mediated interactions. In Chapter VI we consider a nanoparticle based drug delivery platform for targeted, cell specific chemotherapy. In Chapter VII we present prospects for future research: the connection between DNA-mediated colloidal crystallization and jamming, and the inverse problem in self-assembly.

DNA assembly with error correction on a droplet digital microfluidics platform.

PubMed

Khilko, Yuliya; Weyman, Philip D; Glass, John I; Adams, Mark D; McNeil, Melanie A; Griffin, Peter B

2018-06-01

Custom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform. We optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 μL. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl 2 , Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb - 1 . We demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization.

Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten.

PubMed

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.

Platforms.

PubMed

Josko, Deborah

2014-01-01

The advent of DNA sequencing technologies and the various applications that can be performed will have a dramatic effect on medicine and healthcare in the near future. There are several DNA sequencing platforms available on the market for research and clinical use. Based on the medical laboratory scientist or researcher's needs and taking into consideration laboratory space and budget, one can chose which platform will be beneficial to their institution and their patient population. Although some of the instrument costs seem high, diagnosing a patient quickly and accurately will save hospitals money with fewer hospital stays and targeted treatment based on an individual's genetic make-up. By determining the type of disease an individual has, based on the mutations present or having the ability to prescribe the appropriate antimicrobials based on the knowledge of the organism's resistance patterns, the clinician will be better able to treat and diagnose a patient which ultimately will improve patient outcomes and prognosis.

Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers

PubMed Central

Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M.; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

2016-01-01

Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage. TABSAT is freely available under a GNU General Public License version 3.0 (GPLv3) at https://github.com/tadkeys/tabsat/ and http://demo.platomics.com/. PMID:27467908

Toward a mtDNA locus-specific mutation database using the LOVD platform.

PubMed

Elson, Joanna L; Sweeney, Mary G; Procaccio, Vincent; Yarham, John W; Salas, Antonio; Kong, Qing-Peng; van der Westhuizen, Francois H; Pitceathly, Robert D S; Thorburn, David R; Lott, Marie T; Wallace, Douglas C; Taylor, Robert W; McFarland, Robert

2012-09-01

The Human Variome Project (HVP) is a global effort to collect and curate all human genetic variation affecting health. Mutations of mitochondrial DNA (mtDNA) are an important cause of neurogenetic disease in humans; however, identification of the pathogenic mutations responsible can be problematic. In this article, we provide explanations as to why and suggest how such difficulties might be overcome. We put forward a case in support of a new Locus Specific Mutation Database (LSDB) implemented using the Leiden Open-source Variation Database (LOVD) system that will not only list primary mutations, but also present the evidence supporting their role in disease. Critically, we feel that this new database should have the capacity to store information on the observed phenotypes alongside the genetic variation, thereby facilitating our understanding of the complex and variable presentation of mtDNA disease. LOVD supports fast queries of both seen and hidden data and allows storage of sequence variants from high-throughput sequence analysis. The LOVD platform will allow construction of a secure mtDNA database; one that can fully utilize currently available data, as well as that being generated by high-throughput sequencing, to link genotype with phenotype enhancing our understanding of mitochondrial disease, with a view to providing better prognostic information. © 2012 Wiley Periodicals, Inc.

Toward a mtDNA Locus-Specific Mutation Database Using the LOVD Platform

PubMed Central

Elson, Joanna L.; Sweeney, Mary G.; Procaccio, Vincent; Yarham, John W.; Salas, Antonio; Kong, Qing-Peng; van der Westhuizen, Francois H.; Pitceathly, Robert D.S.; Thorburn, David R.; Lott, Marie T.; Wallace, Douglas C.; Taylor, Robert W.; McFarland, Robert

2015-01-01

The Human Variome Project (HVP) is a global effort to collect and curate all human genetic variation affecting health. Mutations of mitochondrial DNA (mtDNA) are an important cause of neurogenetic disease in humans; however, identification of the pathogenic mutations responsible can be problematic. In this article, we provide explanations as to why and suggest how such difficulties might be overcome. We put forward a case in support of a new Locus Specific Mutation Database (LSDB) implemented using the Leiden Open-source Variation Database (LOVD) system that will not only list primary mutations, but also present the evidence supporting their role in disease. Critically, we feel that this new database should have the capacity to store information on the observed phenotypes alongside the genetic variation, thereby facilitating our understanding of the complex and variable presentation of mtDNA disease. LOVD supports fast queries of both seen and hidden data and allows storage of sequence variants from high-throughput sequence analysis. The LOVD platform will allow construction of a secure mtDNA database; one that can fully utilize currently available data, as well as that being generated by high-throughput sequencing, to link genotype with phenotype enhancing our understanding of mitochondrial disease, with a view to providing better prognostic information. PMID:22581690

[Review of Second Generation Sequencing and Its Application in Forensic Genetics].

PubMed

Zhang, S H; Bian, Y N; Zhao, Q; Li, C T

2016-08-01

The rapid development of second generation sequencing （SGS） within the past few years has led to the increasement of data throughput and read length while at the same time brought down substantially the sequencing cost. This made new breakthrough in the area of biology and ushered the forensic genetics into a new era. Based on the history of sequencing application in forensic genetics, this paper reviews the importance of sequencing technologies for genetic marker detection. The application status and potential of SGS in forensic genetics are discussed based on the already explored SGS platforms of Roche, Illumina and Life Technologies. With these platforms, DNA markers （SNP, STR）, RNA markers （mRNA, microRNA） and whole mtDNA can be sequenced. However, development and validation of application kits, maturation of analysis software, connection to the existing databases and the possible ethical issues occurred with big data will be the key factors that determine whether this technology can substitute or supplement PCR-CE, the mature technology, and be widely used for cases detection. Copyright© by the Editorial Department of Journal of Forensic Medicine.

A versatile phenotyping system and analytics platform reveals diverse temporal responses to water availability in Setaria

USDA-ARS?s Scientific Manuscript database

With rapid advances in DNA sequencing, phenotyping has become the rate-limiting step in using large-scale genomic data to understand and improve agricultural crops. Here, the Bellwether Phenotyping platform for controlled-environment plant growth and automated, multimodal phenotyping is described. T...

An end-to-end workflow for engineering of biological networks from high-level specifications.

PubMed

Beal, Jacob; Weiss, Ron; Densmore, Douglas; Adler, Aaron; Appleton, Evan; Babb, Jonathan; Bhatia, Swapnil; Davidsohn, Noah; Haddock, Traci; Loyall, Joseph; Schantz, Richard; Vasilev, Viktor; Yaman, Fusun

2012-08-17

We present a workflow for the design and production of biological networks from high-level program specifications. The workflow is based on a sequence of intermediate models that incrementally translate high-level specifications into DNA samples that implement them. We identify algorithms for translating between adjacent models and implement them as a set of software tools, organized into a four-stage toolchain: Specification, Compilation, Part Assignment, and Assembly. The specification stage begins with a Boolean logic computation specified in the Proto programming language. The compilation stage uses a library of network motifs and cellular platforms, also specified in Proto, to transform the program into an optimized Abstract Genetic Regulatory Network (AGRN) that implements the programmed behavior. The part assignment stage assigns DNA parts to the AGRN, drawing the parts from a database for the target cellular platform, to create a DNA sequence implementing the AGRN. Finally, the assembly stage computes an optimized assembly plan to create the DNA sequence from available part samples, yielding a protocol for producing a sample of engineered plasmids with robotics assistance. Our workflow is the first to automate the production of biological networks from a high-level program specification. Furthermore, the workflow's modular design allows the same program to be realized on different cellular platforms simply by swapping workflow configurations. We validated our workflow by specifying a small-molecule sensor-reporter program and verifying the resulting plasmids in both HEK 293 mammalian cells and in E. coli bacterial cells.

Autonomous replication of nucleic acids by polymerization/nicking enzyme/DNAzyme cascades for the amplified detection of DNA and the aptamer-cocaine complex.

PubMed

Wang, Fuan; Freage, Lina; Orbach, Ron; Willner, Itamar

2013-09-03

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg(2+)-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg(2+)-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM).

An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

PubMed

Huang, Shu-Hong; Chang, Yu-Shin; Juang, Jyh-Ming Jimmy; Chang, Kai-Wei; Tsai, Mong-Hsun; Lu, Tzu-Pin; Lai, Liang-Chuan; Chuang, Eric Y; Huang, Nien-Tsu

2018-03-12

In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection

NASA Astrophysics Data System (ADS)

Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih

2017-04-01

Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5‧ and 3‧ termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

Googling DNA sequences on the World Wide Web.

PubMed

Hajibabaei, Mehrdad; Singer, Gregory A C

2009-11-10

New web-based technologies provide an excellent opportunity for sharing and accessing information and using web as a platform for interaction and collaboration. Although several specialized tools are available for analyzing DNA sequence information, conventional web-based tools have not been utilized for bioinformatics applications. We have developed a novel algorithm and implemented it for searching species-specific genomic sequences, DNA barcodes, by using popular web-based methods such as Google. We developed an alignment independent character based algorithm based on dividing a sequence library (DNA barcodes) and query sequence to words. The actual search is conducted by conventional search tools such as freely available Google Desktop Search. We implemented our algorithm in two exemplar packages. We developed pre and post-processing software to provide customized input and output services, respectively. Our analysis of all publicly available DNA barcode sequences shows a high accuracy as well as rapid results. Our method makes use of conventional web-based technologies for specialized genetic data. It provides a robust and efficient solution for sequence search on the web. The integration of our search method for large-scale sequence libraries such as DNA barcodes provides an excellent web-based tool for accessing this information and linking it to other available categories of information on the web.

DNA polymorphism sensitive impedimetric detection on gold-nanoislands modified electrodes.

PubMed

Bonanni, Alessandra; Pividori, Maria Isabel; del Valle, Manel

2015-05-01

Nanocomposite materials are being increasingly used in biosensing applications as they can significantly improve biosensor performance. Here we report the use of a novel impedimetric genosensor based on gold nanoparticles graphite-epoxy nanocomposite (nanoAu-GEC) for the detection of triple base mutation deletion in a cystic-fibrosis (CF) related human DNA sequence. The developed platform consists of chemisorbing gold nano-islands surrounded by rigid, non-chemisorbing, and conducting graphite-epoxy composite. The ratio of the gold nanoparticles in the composite was carefully optimized by electrochemical and microscopy studies. Such platform allows the very fast and stable thiol immobilization of DNA probes on the gold islands, thus minimizing the steric and electrostatic repulsion among the DNA probes and improving the detection of DNA polymorphism down to 2.25fmol by using electrochemical impedance spectroscopy. These findings are very important in order to develop new and renewable platforms to be used in point-of-care devices for the detection of biomolecules. Copyright © 2015 Elsevier B.V. All rights reserved.

Carbon nanostructures as immobilization platform for DNA: A review on current progress in electrochemical DNA sensors.

PubMed

Rasheed, P Abdul; Sandhyarani, N

2017-11-15

Development of a sensitive, specific and cost-effective DNA detection method is motivated by increasing demand for the early stage diagnosis of genetic diseases. Recent developments in the design and fabrication of efficient sensor platforms based on nanostructures make the highly sensitive sensors which could indicate very low detection limit to the level of few molecules, a realistic possibility. Electrochemical detection methods are widely used in DNA diagnostics as it provide simple, accurate and inexpensive platform for DNA detection. In addition, the electrochemical DNA sensors provide direct electronic signal without the use of expensive signal transduction equipment and facilitates the immobilization of single stranded DNA (ssDNA) probe sequences on a wide variety of electrode substrates. It has been found that a range of nanomaterials such as metal nanoparticles (MNPs), carbon based nanomaterials, quantum dots (QDs), magnetic nanoparticles and polymeric NPs have been introduced in the sensor design to enhance the sensing performance of electrochemical DNA sensor. In this review, we discuss recent progress in the design and fabrication of efficient electrochemical genosensors based on carbon nanostructures such as carbon nanotubes, graphene, graphene oxide and nanodiamonds. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid and Easy Protocol for Quantification of Next-Generation Sequencing Libraries.

PubMed

Hawkins, Steve F C; Guest, Paul C

2018-01-01

The emergence of next-generation sequencing (NGS) over the last 10 years has increased the efficiency of DNA sequencing in terms of speed, ease, and price. However, the exact quantification of a NGS library is crucial in order to obtain good data on sequencing platforms developed by the current market leader Illumina. Different approaches for DNA quantification are available currently and the most commonly used are based on analysis of the physical properties of the DNA through spectrophotometric or fluorometric methods. Although these methods are technically simple, they do not allow exact quantification as can be achieved using a real-time quantitative PCR (qPCR) approach. A qPCR protocol for DNA quantification with applications in NGS library preparation studies is presented here. This can be applied in various fields of study such as medical disorders resulting from nutritional programming disturbances.

Accounting for uncertainty in DNA sequencing data.

PubMed

O'Rawe, Jason A; Ferson, Scott; Lyon, Gholson J

2015-02-01

Science is defined in part by an honest exposition of the uncertainties that arise in measurements and propagate through calculations and inferences, so that the reliabilities of its conclusions are made apparent. The recent rapid development of high-throughput DNA sequencing technologies has dramatically increased the number of measurements made at the biochemical and molecular level. These data come from many different DNA-sequencing technologies, each with their own platform-specific errors and biases, which vary widely. Several statistical studies have tried to measure error rates for basic determinations, but there are no general schemes to project these uncertainties so as to assess the surety of the conclusions drawn about genetic, epigenetic, and more general biological questions. We review here the state of uncertainty quantification in DNA sequencing applications, describe sources of error, and propose methods that can be used for accounting and propagating these errors and their uncertainties through subsequent calculations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Base resolution methylome profiling: considerations in platform selection, data preprocessing and analysis

PubMed Central

Sun, Zhifu; Cunningham, Julie; Slager, Susan; Kocher, Jean-Pierre

2015-01-01

Bisulfite treatment-based methylation microarray (mainly Illumina 450K Infinium array) and next-generation sequencing (reduced representation bisulfite sequencing, Agilent SureSelect Human Methyl-Seq, NimbleGen SeqCap Epi CpGiant or whole-genome bisulfite sequencing) are commonly used for base resolution DNA methylome research. Although multiple tools and methods have been developed and used for the data preprocessing and analysis, confusions remains for these platforms including how and whether the 450k array should be normalized; which platform should be used to better fit researchers’ needs; and which statistical models would be more appropriate for differential methylation analysis. This review presents the commonly used platforms and compares the pros and cons of each in methylome profiling. We then discuss approaches to study design, data normalization, bias correction and model selection for differentially methylated individual CpGs and regions. PMID:26366945

Low-Energy Electron-Induced Strand Breaks in Telomere-Derived DNA Sequences-Influence of DNA Sequence and Topology.

PubMed

Rackwitz, Jenny; Bald, Ilko

2018-03-26

During cancer radiation therapy high-energy radiation is used to reduce tumour tissue. The irradiation produces a shower of secondary low-energy (<20 eV) electrons, which are able to damage DNA very efficiently by dissociative electron attachment. Recently, it was suggested that low-energy electron-induced DNA strand breaks strongly depend on the specific DNA sequence with a high sensitivity of G-rich sequences. Here, we use DNA origami platforms to expose G-rich telomere sequences to low-energy (8.8 eV) electrons to determine absolute cross sections for strand breakage and to study the influence of sequence modifications and topology of telomeric DNA on the strand breakage. We find that the telomeric DNA 5'-(TTA GGG) 2 is more sensitive to low-energy electrons than an intermixed sequence 5'-(TGT GTG A) 2 confirming the unique electronic properties resulting from G-stacking. With increasing length of the oligonucleotide (i.e., going from 5'-(GGG ATT) 2 to 5'-(GGG ATT) 4 ), both the variety of topology and the electron-induced strand break cross sections increase. Addition of K + ions decreases the strand break cross section for all sequences that are able to fold G-quadruplexes or G-intermediates, whereas the strand break cross section for the intermixed sequence remains unchanged. These results indicate that telomeric DNA is rather sensitive towards low-energy electron-induced strand breakage suggesting significant telomere shortening that can also occur during cancer radiation therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A remark on copy number variation detection methods.

PubMed

Li, Shuo; Dou, Xialiang; Gao, Ruiqi; Ge, Xinzhou; Qian, Minping; Wan, Lin

2018-01-01

Copy number variations (CNVs) are gain and loss of DNA sequence of a genome. High throughput platforms such as microarrays and next generation sequencing technologies (NGS) have been applied for genome wide copy number losses. Although progress has been made in both approaches, the accuracy and consistency of CNV calling from the two platforms remain in dispute. In this study, we perform a deep analysis on copy number losses on 254 human DNA samples, which have both SNP microarray data and NGS data publicly available from Hapmap Project and 1000 Genomes Project respectively. We show that the copy number losses reported from Hapmap Project and 1000 Genome Project only have < 30% overlap, while these reports are required to have cross-platform (e.g. PCR, microarray and high-throughput sequencing) experimental supporting by their corresponding projects, even though state-of-art calling methods were employed. On the other hand, copy number losses are found directly from HapMap microarray data by an accurate algorithm, i.e. CNVhac, almost all of which have lower read mapping depth in NGS data; furthermore, 88% of which can be supported by the sequences with breakpoint in NGS data. Our results suggest the ability of microarray calling CNVs and the possible introduction of false negatives from the unessential requirement of the additional cross-platform supporting. The inconsistency of CNV reports from Hapmap Project and 1000 Genomes Project might result from the inadequate information containing in microarray data, the inconsistent detection criteria, or the filtration effect of cross-platform supporting. The statistical test on CNVs called from CNVhac show that the microarray data can offer reliable CNV reports, and majority of CNV candidates can be confirmed by raw sequences. Therefore, the CNV candidates given by a good caller could be highly reliable without cross-platform supporting, so additional experimental information should be applied in need instead of necessarily.

Detection and interrogation of biomolecules via nanoscale probes: From fundamental physics to DNA sequencing

NASA Astrophysics Data System (ADS)

Zwolak, Michael

2013-03-01

A rapid and low-cost method to sequence DNA would revolutionize personalized medicine, where genetic information is used to diagnose, treat, and prevent diseases. There is a longstanding interest in nanopores as a platform for rapid interrogation of single DNA molecules. I will discuss a sequencing protocol based on the measurement of transverse electronic currents during the translocation of single-stranded DNA through nanopores. Using molecular dynamics simulations coupled to quantum mechanical calculations of the tunneling current, I will show that the DNA nucleotides are predicted to have distinguishable electronic signatures in experimentally realizable systems. Several recent experiments support our theoretical predictions. In addition to their possible impact in medicine and biology, the above methods offer ideal test beds to study open scientific issues in the relatively unexplored area at the interface between solids, liquids, and biomolecules at the nanometer length scale. http://mike.zwolak.org

Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform.

PubMed

Rutvisuttinunt, Wiriya; Chinnawirotpisan, Piyawan; Simasathien, Sriluck; Shrestha, Sanjaya K; Yoon, In-Kyu; Klungthong, Chonticha; Fernandez, Stefan

2013-11-01

Active global surveillance and characterization of influenza viruses are essential for better preparation against possible pandemic events. Obtaining comprehensive information about the influenza genome can improve our understanding of the evolution of influenza viruses and emergence of new strains, and improve the accuracy when designing preventive vaccines. This study investigated the use of deep sequencing by the next-generation sequencing (NGS) Illumina MiSeq Platform to obtain complete genome sequence information from influenza virus isolates. The influenza virus isolates were cultured from 6 respiratory acute clinical specimens collected in Thailand and Nepal. DNA libraries obtained from each viral isolate were mixed and all were sequenced simultaneously. Total information of 2.6 Gbases was obtained from a 455±14 K/mm2 density with 95.76% (8,571,655/8,950,724 clusters) of the clusters passing quality control (QC) filters. Approximately 93.7% of all sequences from Read1 and 83.5% from Read2 contained high quality sequences that were ≥Q30, a base calling QC score standard. Alignments analysis identified three seasonal influenza A H3N2 strains, one 2009 pandemic influenza A H1N1 strain and two influenza B strains. The nearly entire genomes of all six virus isolates yielded equal or greater than 600-fold sequence coverage depth. MiSeq Platform identified seasonal influenza A H3N2, 2009 pandemic influenza A H1N1and influenza B in the DNA library mixtures efficiently. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Draft genome sequence of Cryptococcus terricola JCM 24523, an oleaginous yeast capable of expressing exogenous DNA

DOE PAGES

Close, Dan; Ojumu, John O.; Zhang, Gui X.

2016-11-03

Cryptococcus terricola JCM 24523 has recently been identified as an oleaginous yeast capable of converting starch into fatty acids. Here, this draft genome sequence provides a platform for elucidating its fatty acid production potential and supporting comparisons with other oleaginous species.

Draft genome sequence of Cryptococcus terricola JCM 24523, an oleaginous yeast capable of expressing exogenous DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Close, Dan; Ojumu, John O.; Zhang, Gui X.

Cryptococcus terricola JCM 24523 has recently been identified as an oleaginous yeast capable of converting starch into fatty acids. Here, this draft genome sequence provides a platform for elucidating its fatty acid production potential and supporting comparisons with other oleaginous species.

Portable and Error-Free DNA-Based Data Storage.

PubMed

Yazdi, S M Hossein Tabatabaei; Gabrys, Ryan; Milenkovic, Olgica

2017-07-10

DNA-based data storage is an emerging nonvolatile memory technology of potentially unprecedented density, durability, and replication efficiency. The basic system implementation steps include synthesizing DNA strings that contain user information and subsequently retrieving them via high-throughput sequencing technologies. Existing architectures enable reading and writing but do not offer random-access and error-free data recovery from low-cost, portable devices, which is crucial for making the storage technology competitive with classical recorders. Here we show for the first time that a portable, random-access platform may be implemented in practice using nanopore sequencers. The novelty of our approach is to design an integrated processing pipeline that encodes data to avoid costly synthesis and sequencing errors, enables random access through addressing, and leverages efficient portable sequencing via new iterative alignment and deletion error-correcting codes. Our work represents the only known random access DNA-based data storage system that uses error-prone nanopore sequencers, while still producing error-free readouts with the highest reported information rate/density. As such, it represents a crucial step towards practical employment of DNA molecules as storage media.

A review of bioinformatic methods for forensic DNA analyses.

PubMed

Liu, Yao-Yuan; Harbison, SallyAnn

2018-03-01

Short tandem repeats, single nucleotide polymorphisms, and whole mitochondrial analyses are three classes of markers which will play an important role in the future of forensic DNA typing. The arrival of massively parallel sequencing platforms in forensic science reveals new information such as insights into the complexity and variability of the markers that were previously unseen, along with amounts of data too immense for analyses by manual means. Along with the sequencing chemistries employed, bioinformatic methods are required to process and interpret this new and extensive data. As more is learnt about the use of these new technologies for forensic applications, development and standardization of efficient, favourable tools for each stage of data processing is being carried out, and faster, more accurate methods that improve on the original approaches have been developed. As forensic laboratories search for the optimal pipeline of tools, sequencer manufacturers have incorporated pipelines into sequencer software to make analyses convenient. This review explores the current state of bioinformatic methods and tools used for the analyses of forensic markers sequenced on the massively parallel sequencing (MPS) platforms currently most widely used. Copyright © 2017 Elsevier B.V. All rights reserved.

Single helically folded aromatic oligoamides that mimic the charge surface of double-stranded B-DNA

NASA Astrophysics Data System (ADS)

Ziach, Krzysztof; Chollet, Céline; Parissi, Vincent; Prabhakaran, Panchami; Marchivie, Mathieu; Corvaglia, Valentina; Bose, Partha Pratim; Laxmi-Reddy, Katta; Godde, Frédéric; Schmitter, Jean-Marie; Chaignepain, Stéphane; Pourquier, Philippe; Huc, Ivan

2018-05-01

Numerous essential biomolecular processes require the recognition of DNA surface features by proteins. Molecules mimicking these features could potentially act as decoys and interfere with pharmacologically or therapeutically relevant protein-DNA interactions. Although naturally occurring DNA-mimicking proteins have been described, synthetic tunable molecules that mimic the charge surface of double-stranded DNA are not known. Here, we report the design, synthesis and structural characterization of aromatic oligoamides that fold into single helical conformations and display a double helical array of negatively charged residues in positions that match the phosphate moieties in B-DNA. These molecules were able to inhibit several enzymes possessing non-sequence-selective DNA-binding properties, including topoisomerase 1 and HIV-1 integrase, presumably through specific foldamer-protein interactions, whereas sequence-selective enzymes were not inhibited. Such modular and synthetically accessible DNA mimics provide a versatile platform to design novel inhibitors of protein-DNA interactions.

Denoising DNA deep sequencing data—high-throughput sequencing errors and their correction

PubMed Central

Laehnemann, David; Borkhardt, Arndt

2016-01-01

Characterizing the errors generated by common high-throughput sequencing platforms and telling true genetic variation from technical artefacts are two interdependent steps, essential to many analyses such as single nucleotide variant calling, haplotype inference, sequence assembly and evolutionary studies. Both random and systematic errors can show a specific occurrence profile for each of the six prominent sequencing platforms surveyed here: 454 pyrosequencing, Complete Genomics DNA nanoball sequencing, Illumina sequencing by synthesis, Ion Torrent semiconductor sequencing, Pacific Biosciences single-molecule real-time sequencing and Oxford Nanopore sequencing. There is a large variety of programs available for error removal in sequencing read data, which differ in the error models and statistical techniques they use, the features of the data they analyse, the parameters they determine from them and the data structures and algorithms they use. We highlight the assumptions they make and for which data types these hold, providing guidance which tools to consider for benchmarking with regard to the data properties. While no benchmarking results are included here, such specific benchmarks would greatly inform tool choices and future software development. The development of stand-alone error correctors, as well as single nucleotide variant and haplotype callers, could also benefit from using more of the knowledge about error profiles and from (re)combining ideas from the existing approaches presented here. PMID:26026159

Methods for open innovation on a genome-design platform associating scientific, commercial, and educational communities in synthetic biology.

PubMed

Toyoda, Tetsuro

2011-01-01

Synthetic biology requires both engineering efficiency and compliance with safety guidelines and ethics. Focusing on the rational construction of biological systems based on engineering principles, synthetic biology depends on a genome-design platform to explore the combinations of multiple biological components or BIO bricks for quickly producing innovative devices. This chapter explains the differences among various platform models and details a methodology for promoting open innovation within the scope of the statutory exemption of patent laws. The detailed platform adopts a centralized evaluation model (CEM), computer-aided design (CAD) bricks, and a freemium model. It is also important for the platform to support the legal aspects of copyrights as well as patent and safety guidelines because intellectual work including DNA sequences designed rationally by human intelligence is basically copyrightable. An informational platform with high traceability, transparency, auditability, and security is required for copyright proof, safety compliance, and incentive management for open innovation in synthetic biology. GenoCon, which we have organized and explained here, is a competition-styled, open-innovation method involving worldwide participants from scientific, commercial, and educational communities that aims to improve the designs of genomic sequences that confer a desired function on an organism. Using only a Web browser, a participating contributor proposes a design expressed with CAD bricks that generate a relevant DNA sequence, which is then experimentally and intensively evaluated by the GenoCon organizers. The CAD bricks that comprise programs and databases as a Semantic Web are developed, executed, shared, reused, and well stocked on the secure Semantic Web platform called the Scientists' Networking System or SciNetS/SciNeS, based on which a CEM research center for synthetic biology and open innovation should be established. Copyright © 2011 Elsevier Inc. All rights reserved.

ChIP-chip versus ChIP-seq: Lessons for experimental design and data analysis

PubMed Central

2011-01-01

Background Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq) allows genome-wide discovery of protein-DNA interactions such as transcription factor bindings and histone modifications. Previous reports only compared a small number of profiles, and little has been done to compare histone modification profiles generated by the two technologies or to assess the impact of input DNA libraries in ChIP-seq analysis. Here, we performed a systematic analysis of a modENCODE dataset consisting of 31 pairs of ChIP-chip/ChIP-seq profiles of the coactivator CBP, RNA polymerase II (RNA PolII), and six histone modifications across four developmental stages of Drosophila melanogaster. Results Both technologies produce highly reproducible profiles within each platform, ChIP-seq generally produces profiles with a better signal-to-noise ratio, and allows detection of more peaks and narrower peaks. The set of peaks identified by the two technologies can be significantly different, but the extent to which they differ varies depending on the factor and the analysis algorithm. Importantly, we found that there is a significant variation among multiple sequencing profiles of input DNA libraries and that this variation most likely arises from both differences in experimental condition and sequencing depth. We further show that using an inappropriate input DNA profile can impact the average signal profiles around genomic features and peak calling results, highlighting the importance of having high quality input DNA data for normalization in ChIP-seq analysis. Conclusions Our findings highlight the biases present in each of the platforms, show the variability that can arise from both technology and analysis methods, and emphasize the importance of obtaining high quality and deeply sequenced input DNA libraries for ChIP-seq analysis. PMID:21356108

Comparison of performance of three commercial platforms for warfarin sensitivity genotyping.

PubMed

Babic, Nikolina; Haverfield, Eden V; Burrus, Julie A; Lozada, Anthony; Das, Soma; Yeo, Kiang-Teck J

2009-08-01

We performed a 3-way comparison on the Osmetech eSensor, AutoGenomics INFINITI, and a real-time PCR method (Paragonx reagents/Stratagene RT-PCR platform) for their FDA-cleared warfarin panels, and additional polymorphisms (CYP2C9*5, *6, and 11 and extended VKORC1 panels) where available. One hundred de-identified DNA samples were used in this IRB-approved study. Accuracy was determined by comparison of genotyping results across three platforms. Any discrepancy was resolved by bi-directional sequencing. The CYP4F2 on Osmetech was validated by bi-directional sequencing. Accuracies for CYP2C9*2 and *3 were 100% for all 3 platforms. VKORC1 3673 genotyping accuracies were 100% on eSensor and 97% on Infiniti. CYP2C9*5, *6 and *11 showed 100% concordance between eSensor and Infiniti. VKORC1 6484 and 9041 variants compared between ParagonDx and Infiniti analyzer were 100% (6484) and 99% (9041) concordant. CYP4F2 was 100% concordant with sequencing results. The time required to generate the results from automated DNA extraction-to-result was approximately 8h on Infiniti, and 4h on eSensor and ParagonDx, respectively. Overall, we observed excellent CYP2C9*2 and *3 genotyping accuracy for all three platforms. For VKORC1 3673 genotyping, eSensor demonstrated a slightly higher accuracy than the Infiniti, and CYP4F2 on Osmetech was 100% accurate.

Initial steps towards a production platform for DNA sequence analysis on the grid.

PubMed

Luyf, Angela C M; van Schaik, Barbera D C; de Vries, Michel; Baas, Frank; van Kampen, Antoine H C; Olabarriaga, Silvia D

2010-12-14

Bioinformatics is confronted with a new data explosion due to the availability of high throughput DNA sequencers. Data storage and analysis becomes a problem on local servers, and therefore it is needed to switch to other IT infrastructures. Grid and workflow technology can help to handle the data more efficiently, as well as facilitate collaborations. However, interfaces to grids are often unfriendly to novice users. In this study we reused a platform that was developed in the VL-e project for the analysis of medical images. Data transfer, workflow execution and job monitoring are operated from one graphical interface. We developed workflows for two sequence alignment tools (BLAST and BLAT) as a proof of concept. The analysis time was significantly reduced. All workflows and executables are available for the members of the Dutch Life Science Grid and the VL-e Medical virtual organizations All components are open source and can be transported to other grid infrastructures. The availability of in-house expertise and tools facilitates the usage of grid resources by new users. Our first results indicate that this is a practical, powerful and scalable solution to address the capacity and collaboration issues raised by the deployment of next generation sequencers. We currently adopt this methodology on a daily basis for DNA sequencing and other applications. More information and source code is available via http://www.bioinformaticslaboratory.nl/

A parallel and sensitive software tool for methylation analysis on multicore platforms.

PubMed

Tárraga, Joaquín; Pérez, Mariano; Orduña, Juan M; Duato, José; Medina, Ignacio; Dopazo, Joaquín

2015-10-01

DNA methylation analysis suffers from very long processing time, as the advent of Next-Generation Sequencers has shifted the bottleneck of genomic studies from the sequencers that obtain the DNA samples to the software that performs the analysis of these samples. The existing software for methylation analysis does not seem to scale efficiently neither with the size of the dataset nor with the length of the reads to be analyzed. As it is expected that the sequencers will provide longer and longer reads in the near future, efficient and scalable methylation software should be developed. We present a new software tool, called HPG-Methyl, which efficiently maps bisulphite sequencing reads on DNA, analyzing DNA methylation. The strategy used by this software consists of leveraging the speed of the Burrows-Wheeler Transform to map a large number of DNA fragments (reads) rapidly, as well as the accuracy of the Smith-Waterman algorithm, which is exclusively employed to deal with the most ambiguous and shortest reads. Experimental results on platforms with Intel multicore processors show that HPG-Methyl significantly outperforms in both execution time and sensitivity state-of-the-art software such as Bismark, BS-Seeker or BSMAP, particularly for long bisulphite reads. Software in the form of C libraries and functions, together with instructions to compile and execute this software. Available by sftp to anonymous@clariano.uv.es (password 'anonymous'). juan.orduna@uv.es or jdopazo@cipf.es. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Identification of Genomic Insertion and Flanking Sequence of G2-EPSPS and GAT Transgenes in Soybean Using Whole Genome Sequencing Method.

PubMed

Guo, Bingfu; Guo, Yong; Hong, Huilong; Qiu, Li-Juan

2016-01-01

Molecular characterization of sequence flanking exogenous fragment insertion is essential for safety assessment and labeling of genetically modified organism (GMO). In this study, the T-DNA insertion sites and flanking sequences were identified in two newly developed transgenic glyphosate-tolerant soybeans GE-J16 and ZH10-6 based on whole genome sequencing (WGS) method. More than 22.4 Gb sequence data (∼21 × coverage) for each line was generated on Illumina HiSeq 2500 platform. The junction reads mapped to boundaries of T-DNA and flanking sequences in these two events were identified by comparing all sequencing reads with soybean reference genome and sequence of transgenic vector. The putative insertion loci and flanking sequences were further confirmed by PCR amplification, Sanger sequencing, and co-segregation analysis. All these analyses supported that exogenous T-DNA fragments were integrated in positions of Chr19: 50543767-50543792 and Chr17: 7980527-7980541 in these two transgenic lines. Identification of genomic insertion sites of G2-EPSPS and GAT transgenes will facilitate the utilization of their glyphosate-tolerant traits in soybean breeding program. These results also demonstrated that WGS was a cost-effective and rapid method for identifying sites of T-DNA insertions and flanking sequences in soybean.

DNA sequencing using polymerase substrate-binding kinetics

PubMed Central

Previte, Michael John Robert; Zhou, Chunhong; Kellinger, Matthew; Pantoja, Rigo; Chen, Cheng-Yao; Shi, Jin; Wang, BeiBei; Kia, Amirali; Etchin, Sergey; Vieceli, John; Nikoomanzar, Ali; Bomati, Erin; Gloeckner, Christian; Ronaghi, Mostafa; He, Molly Min

2015-01-01

Next-generation sequencing (NGS) has transformed genomic research by decreasing the cost of sequencing. However, whole-genome sequencing is still costly and complex for diagnostics purposes. In the clinical space, targeted sequencing has the advantage of allowing researchers to focus on specific genes of interest. Routine clinical use of targeted NGS mandates inexpensive instruments, fast turnaround time and an integrated and robust workflow. Here we demonstrate a version of the Sequencing by Synthesis (SBS) chemistry that potentially can become a preferred targeted sequencing method in the clinical space. This sequencing chemistry uses natural nucleotides and is based on real-time recording of the differential polymerase/DNA-binding kinetics in the presence of correct or mismatch nucleotides. This ensemble SBS chemistry has been implemented on an existing Illumina sequencing platform with integrated cluster amplification. We discuss the advantages of this sequencing chemistry for targeted sequencing as well as its limitations for other applications. PMID:25612848

Current status and future perspectives on molecular and serological methods in diagnostic mycology.

PubMed

Lau, Anna; Chen, Sharon; Sleiman, Sue; Sorrell, Tania

2009-11-01

Invasive fungal infections are an important cause of infectious morbidity. Nonculture-based methods are increasingly used for rapid, accurate diagnosis to improve patient outcomes. New and existing DNA amplification platforms have high sensitivity and specificity for direct detection and identification of fungi in clinical specimens. Since laboratories are increasingly reliant on DNA sequencing for fungal identification, measures to improve sequence interpretation should support validation of reference isolates and quality control in public gene repositories. Novel technologies (e.g., isothermal and PNA FISH methods), platforms enabling high-throughput analyses (e.g., DNA microarrays and Luminex xMAP) and/or commercial PCR assays warrant further evaluation for routine diagnostic use. Notwithstanding the advantages of molecular tests, serological assays remain clinically useful for patient management. The serum Aspergillus galactomannan test has been incorporated into diagnostic algorithms of invasive aspergillosis. Both the galactomannan and the serum beta-D-glucan test have value for diagnosing infection and monitoring therapeutic response.

Multiple advanced logic gates made of DNA-Ag nanocluster and the application for intelligent detection of pathogenic bacterial genes.

PubMed

Lin, Xiaodong; Liu, Yaqing; Deng, Jiankang; Lyu, Yanlong; Qian, Pengcheng; Li, Yunfei; Wang, Shuo

2018-02-21

The integration of multiple DNA logic gates on a universal platform to implement advance logic functions is a critical challenge for DNA computing. Herein, a straightforward and powerful strategy in which a guanine-rich DNA sequence lighting up a silver nanocluster and fluorophore was developed to construct a library of logic gates on a simple DNA-templated silver nanoclusters (DNA-AgNCs) platform. This library included basic logic gates, YES, AND, OR, INHIBIT, and XOR, which were further integrated into complex logic circuits to implement diverse advanced arithmetic/non-arithmetic functions including half-adder, half-subtractor, multiplexer, and demultiplexer. Under UV irradiation, all the logic functions could be instantly visualized, confirming an excellent repeatability. The logic operations were entirely based on DNA hybridization in an enzyme-free and label-free condition, avoiding waste accumulation and reducing cost consumption. Interestingly, a DNA-AgNCs-based multiplexer was, for the first time, used as an intelligent biosensor to identify pathogenic genes, E. coli and S. aureus genes, with a high sensitivity. The investigation provides a prototype for the wireless integration of multiple devices on even the simplest single-strand DNA platform to perform diverse complex functions in a straightforward and cost-effective way.

An Efficient Method for Electroporation of Small Interfering RNAs into ENCODE Project Tier 1 GM12878 and K562 Cell Lines.

PubMed

Muller, Ryan Y; Hammond, Ming C; Rio, Donald C; Lee, Yeon J

2015-12-01

The Encyclopedia of DNA Elements (ENCODE) Project aims to identify all functional sequence elements in the human genome sequence by use of high-throughput DNA/cDNA sequencing approaches. To aid the standardization, comparison, and integration of data sets produced from different technologies and platforms, the ENCODE Consortium selected several standard human cell lines to be used by the ENCODE Projects. The Tier 1 ENCODE cell lines include GM12878, K562, and H1 human embryonic stem cell lines. GM12878 is a lymphoblastoid cell line, transformed with the Epstein-Barr virus, that was selected by the International HapMap Project for whole genome and transcriptome sequencing by use of the Illumina platform. K562 is an immortalized myelogenous leukemia cell line. The GM12878 cell line is attractive for the ENCODE Projects, as it offers potential synergy with the International HapMap Project. Despite the vast amount of sequencing data available on the GM12878 cell line through the ENCODE Project, including transcriptome, chromatin immunoprecipitation-sequencing for histone marks, and transcription factors, no small interfering siRNA-mediated knockdown studies have been performed in the GM12878 cell line, as cationic lipid-mediated transfection methods are inefficient for lymphoid cell lines. Here, we present an efficient and reproducible method for transfection of a variety of siRNAs into the GM12878 and K562 cell lines, which subsequently results in targeted protein depletion.

From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes

PubMed Central

2014-01-01

Background Next-generation DNA sequencing (NGS) technologies have made huge impacts in many fields of biological research, but especially in evolutionary biology. One area where NGS has shown potential is for high-throughput sequencing of complete mtDNA genomes (of humans and other animals). Despite the increasing use of NGS technologies and a better appreciation of their importance in answering biological questions, there remain significant obstacles to the successful implementation of NGS-based projects, especially for new users. Results Here we present an ‘A to Z’ protocol for obtaining complete human mitochondrial (mtDNA) genomes – from DNA extraction to consensus sequence. Although designed for use on humans, this protocol could also be used to sequence small, organellar genomes from other species, and also nuclear loci. This protocol includes DNA extraction, PCR amplification, fragmentation of PCR products, barcoding of fragments, sequencing using the 454 GS FLX platform, and a complete bioinformatics pipeline (primer removal, reference-based mapping, output of coverage plots and SNP calling). Conclusions All steps in this protocol are designed to be straightforward to implement, especially for researchers who are undertaking next-generation sequencing for the first time. The molecular steps are scalable to large numbers (hundreds) of individuals and all steps post-DNA extraction can be carried out in 96-well plate format. Also, the protocol has been assembled so that individual ‘modules’ can be swapped out to suit available resources. PMID:24460871

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

PubMed

Boltaña, Sebastian; Castellana, Barbara; Goetz, Giles; Tort, Lluis; Teles, Mariana; Mulero, Victor; Novoa, Beatriz; Figueras, Antonio; Goetz, Frederick W; Gallardo-Escarate, Cristian; Planas, Josep V; Mackenzie, Simon

2017-02-03

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response.

Targeted enrichment strategies for next-generation plant biology

Treesearch

Richard Cronn; Brian J. Knaus; Aaron Liston; Peter J. Maughan; Matthew Parks; John V. Syring; Joshua Udall

2012-01-01

The dramatic advances offered by modem DNA sequencers continue to redefine the limits of what can be accomplished in comparative plant biology. Even with recent achievements, however, plant genomes present obstacles that can make it difficult to execute large-scale population and phylogenetic studies on next-generation sequencing platforms. Factors like large genome...

Illumina MiSeq Sequencing for Preliminary Analysis of Microbiome Causing Primary Endodontic Infections in Egypt

PubMed Central

Azab, Marwa Mohamed; Fayyad, Dalia Mukhtar

2018-01-01

The use of high throughput next generation technologies has allowed more comprehensive analysis than traditional Sanger sequencing. The specific aim of this study was to investigate the microbial diversity of primary endodontic infections using Illumina MiSeq sequencing platform in Egyptian patients. Samples were collected from 19 patients in Suez Canal University Hospital (Endodontic Department) using sterile # 15K file and paper points. DNA was extracted using Mo Bio power soil DNA isolation extraction kit followed by PCR amplification and agarose gel electrophoresis. The microbiome was characterized on the basis of the V3 and V4 hypervariable region of the 16S rRNA gene by using paired-end sequencing on Illumina MiSeq device. MOTHUR software was used in sequence filtration and analysis of sequenced data. A total of 1858 operational taxonomic units at 97% similarity were assigned to 26 phyla, 245 families, and 705 genera. Four main phyla Firmicutes, Bacteroidetes, Proteobacteria, and Synergistetes were predominant in all samples. At genus level, Prevotella, Bacillus, Porphyromonas, Streptococcus, and Bacteroides were the most abundant. Illumina MiSeq platform sequencing can be used to investigate oral microbiome composition of endodontic infections. Elucidating the ecology of endodontic infections is a necessary step in developing effective intracanal antimicrobials. PMID:29849646

A paper-based device for double-stranded DNA detection with Zif268

NASA Astrophysics Data System (ADS)

Zhang, Daohong

2017-05-01

Here, a small analytical device was fabricated on both nitrocellulose membrane and filter paper, for the detection of biotinylated double-stranded DNA (dsDNA) from 1 nM. Zif268 was utilized for capturing the target DNA, which was a zinc finger protein that recognized only a dsDNA with specific sequence. Therefore, this detection platform could be utilized for PCR result detection, with the well-designed primers (interpolate both biotin and Zif268 binding sequence). The result of the assay could be recorded by a camera-phone, and analyzed with software. The whole assay finished within 1 hour. Due to the easy fabrication, operation and disposal of this device, this method can be employed in point-of-care detection or on-site monitoring.

DNATagger, colors for codons.

PubMed

Scherer, N M; Basso, D M

2008-09-16

DNATagger is a web-based tool for coloring and editing DNA, RNA and protein sequences and alignments. It is dedicated to the visualization of protein coding sequences and also protein sequence alignments to facilitate the comprehension of evolutionary processes in sequence analysis. The distinctive feature of DNATagger is the use of codons as informative units for coloring DNA and RNA sequences. The codons are colored according to their corresponding amino acids. It is the first program that colors codons in DNA sequences without being affected by "out-of-frame" gaps of alignments. It can handle single gaps and gaps inside the triplets. The program also provides the possibility to edit the alignments and change color patterns and translation tables. DNATagger is a JavaScript application, following the W3C guidelines, designed to work on standards-compliant web browsers. It therefore requires no installation and is platform independent. The web-based DNATagger is available as free and open source software at http://www.inf.ufrgs.br/~dmbasso/dnatagger/.

ABI Base Recall: Automatic Correction and Ends Trimming of DNA Sequences.

PubMed

Elyazghi, Zakaria; Yazouli, Loubna El; Sadki, Khalid; Radouani, Fouzia

2017-12-01

Automated DNA sequencers produce chromatogram files in ABI format. When viewing chromatograms, some ambiguities are shown at various sites along the DNA sequences, because the program implemented in the sequencing machine and used to call bases cannot always precisely determine the right nucleotide, especially when it is represented by either a broad peak or a set of overlaying peaks. In such cases, a letter other than A, C, G, or T is recorded, most commonly N. Thus, DNA sequencing chromatograms need manual examination: checking for mis-calls and truncating the sequence when errors become too frequent. The purpose of this paper is to develop a program allowing the automatic correction of these ambiguities. This application is a Web-based program powered by Shiny and runs under R platform for an easy exploitation. As a part of the interface, we added the automatic ends clipping option, alignment against reference sequences, and BLAST. To develop and test our tool, we collected several bacterial DNA sequences from different laboratories within Institut Pasteur du Maroc and performed both manual and automatic correction. The comparison between the two methods was carried out. As a result, we note that our program, ABI base recall, accomplishes good correction with a high accuracy. Indeed, it increases the rate of identity and coverage and minimizes the number of mismatches and gaps, hence it provides solution to sequencing ambiguities and saves biologists' time and labor.

Self-Assembled DNA Tetrahedral Scaffolds for the Construction of Electrochemiluminescence Biosensor with Programmable DNA Cyclic Amplification.

PubMed

Feng, Qiu-Mei; Guo, Yue-Hua; Xu, Jing-Juan; Chen, Hong-Yuan

2017-05-24

A novel DNA tetrahedron-structured electrochemiluminescence (ECL) platform for bioanalysis with programmable DNA cyclic amplification was developed. In this work, glucose oxidase (GOD) was labeled to a DNA sequence (S) as functional conjugation (GOD-S), which could hybridize with other DNA sequences (L and P) to form GOD-S:L:P probe. In the presence of target DNA and a help DNA (A), the programmable DNA cyclic amplification was activated and released GOD-S via toehold-mediated strand displacement. Then, the obtained GOD-S was further immobilized on the DNA tetrahedral scaffolds with a pendant capture DNA and Ru(bpy) 3 2+ -conjugated silica nanoparticles (RuSi NPs) decorated on the electrode surface. Thus, the amount of GOD-S assembled on the electrode surface depended on the concentration of target DNA and GOD could catalyze glucose to generate H 2 O 2 in situ. The ECL signal of Ru(bpy) 3 2+ -TPrA system was quenched by the presence of H 2 O 2 . By integrating the programmable DNA cyclic amplification and in situ generating H 2 O 2 as Ru(bpy) 3 2+ ECL quencher, a sensitive DNA tetrahedron-structured ECL sensing platform was proposed for DNA detection. Under optimized conditions, this biosensor showed a wide linear range from 100 aM to 10 pM with a detection limit of 40 aM, indicating a promising application in DNA analysis. Furthermore, by labeling GOD to different recognition elements, the proposed strategy could be used for the detection of various targets. Thus, this programmable cascade amplification strategy not only retains the high selectivity and good capturing efficiency of tetrahedral-decorated electrode surface but also provides potential applications in the construction of ECL biosensor.

The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing.

PubMed

Binladen, Jonas; Gilbert, M Thomas P; Bollback, Jonathan P; Panitz, Frank; Bendixen, Christian; Nielsen, Rasmus; Willerslev, Eske

2007-02-14

The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources. We use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20 DNA Sequencing System (GS20, Roche/454 Life Sciences). Each DNA sequence is subsequently traced back to its individual source through 5'tag-analysis. We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate<0.4%). Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5'primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial analyses, population genetics, and phylogenetics.

DNA methylation assessment from human slow- and fast-twitch skeletal muscle fibers

PubMed Central

Begue, Gwénaëlle; Raue, Ulrika; Jemiolo, Bozena

2017-01-01

A new application of the reduced representation bisulfite sequencing method was developed using low-DNA input to investigate the epigenetic profile of human slow- and fast-twitch skeletal muscle fibers. Successful library construction was completed with as little as 15 ng of DNA, and high-quality sequencing data were obtained with 32 ng of DNA. Analysis identified 143,160 differentially methylated CpG sites across 14,046 genes. In both fiber types, selected genes predominantly expressed in slow or fast fibers were hypomethylated, which was supported by the RNA-sequencing analysis. These are the first fiber type-specific methylation data from human skeletal muscle and provide a unique platform for future research. NEW & NOTEWORTHY This study validates a low-DNA input reduced representation bisulfite sequencing method for human muscle biopsy samples to investigate the methylation patterns at a fiber type-specific level. These are the first fiber type-specific methylation data reported from human skeletal muscle and thus provide initial insight into basal state differences in myosin heavy chain I and IIa muscle fibers among young, healthy men. PMID:28057818

A Versatile Microfluidic Device for Automating Synthetic Biology.

PubMed

Shih, Steve C C; Goyal, Garima; Kim, Peter W; Koutsoubelis, Nicolas; Keasling, Jay D; Adams, Paul D; Hillson, Nathan J; Singh, Anup K

2015-10-16

New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.

A colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA based on silver nanoclusters and unmodified gold nanoparticles

NASA Astrophysics Data System (ADS)

Qu, Fei; Chen, Zeqiu; You, Jinmao; Song, Cuihua

2018-05-01

Human telomere DNA plays a vital role in genome integrity control and carcinogenesis as an indication for extensive cell proliferation. Herein, silver nanoclusters (Ag NCs) templated by polymer and unmodified gold nanoparticles (Au NPs) are designed as a new colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA. Ag NCs can produce the aggregation of Au NPs, so the color of Au NPs changes to blue and the absorption peak moves to 700 nm. While the telomere DNA can protect Au NPs from aggregation, the color turns to red again and the absorption band blue shift. Benefiting from the obvious color change, we can differentiate the length of telomere DNA by naked eyes. As the length of telomere DNA is longer, the variation of color becomes more noticeable. The detection limits of telomere DNA containing 10, 22, 40, 64 bases are estimated to be 1.41, 1.21, 0.23 and 0.22 nM, respectively. On the other hand, when telomere DNA forms G-quadruplex in the presence of K+, or dsDNA with complementary sequence, both G-quadruplex and dsDNA can protect Au NPs better than the unfolded telomere DNA. Hence, a new colorimetric platform for monitoring structure conversion of DNA is established by Ag NCs-Au NPs system, and to prove this type of application, a selective K+ sensor is developed.

Antibody-Mediated Small Molecule Detection Using Programmable DNA-Switches.

PubMed

Rossetti, Marianna; Ippodrino, Rudy; Marini, Bruna; Palleschi, Giuseppe; Porchetta, Alessandro

2018-06-13

The development of rapid, cost-effective, and single-step methods for the detection of small molecules is crucial for improving the quality and efficiency of many applications ranging from life science to environmental analysis. Unfortunately, current methodologies still require multiple complex, time-consuming washing and incubation steps, which limit their applicability. In this work we present a competitive DNA-based platform that makes use of both programmable DNA-switches and antibodies to detect small target molecules. The strategy exploits both the advantages of proximity-based methods and structure-switching DNA-probes. The platform is modular and versatile and it can potentially be applied for the detection of any small target molecule that can be conjugated to a nucleic acid sequence. Here the rational design of programmable DNA-switches is discussed, and the sensitive, rapid, and single-step detection of different environmentally relevant small target molecules is demonstrated.

Detection of Bacterial Pathogens from Broncho-Alveolar Lavage by Next-Generation Sequencing.

PubMed

Leo, Stefano; Gaïa, Nadia; Ruppé, Etienne; Emonet, Stephane; Girard, Myriam; Lazarevic, Vladimir; Schrenzel, Jacques

2017-09-20

The applications of whole-metagenome shotgun sequencing (WMGS) in routine clinical analysis are still limited. A combination of a DNA extraction procedure, sequencing, and bioinformatics tools is essential for the removal of human DNA and for improving bacterial species identification in a timely manner. We tackled these issues with a broncho-alveolar lavage (BAL) sample from an immunocompromised patient who had developed severe chronic pneumonia. We extracted DNA from the BAL sample with protocols based either on sequential lysis of human and bacterial cells or on the mechanical disruption of all cells. Metagenomic libraries were sequenced on Illumina HiSeq platforms. Microbial community composition was determined by k-mer analysis or by mapping to taxonomic markers. Results were compared to those obtained by conventional clinical culture and molecular methods. Compared to mechanical cell disruption, a sequential lysis protocol resulted in a significantly increased proportion of bacterial DNA over human DNA and higher sequence coverage of Mycobacterium abscessus , Corynebacterium jeikeium and Rothia dentocariosa , the bacteria reported by clinical microbiology tests. In addition, we identified anaerobic bacteria not searched for by the clinical laboratory. Our results further support the implementation of WMGS in clinical routine diagnosis for bacterial identification.

Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

PubMed Central

Sanz, Yolanda

2017-01-01

Abstract The miniaturized and portable DNA sequencer MinION™ has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity. In this study, we tested the ability of the MinION™ platform to perform long amplicon sequencing in order to design new approaches to study microbial diversity using a multi-locus approach. After compiling a robust database by parsing and extracting the rrn bacterial region from more than 67000 complete or draft bacterial genomes, we demonstrated that the data obtained during sequencing of the long amplicon in the MinION™ device using R9 and R9.4 chemistries were sufficient to study 2 mock microbial communities in a multiplex manner and to almost completely reconstruct the microbial diversity contained in the HM782D and D6305 mock communities. Although nanopore-based sequencing produces reads with lower per-base accuracy compared with other platforms, we presented a novel approach consisting of multi-locus and long amplicon sequencing using the MinION™ MkIb DNA sequencer and R9 and R9.4 chemistries that help to overcome the main disadvantage of this portable sequencing platform. Furthermore, the nanopore sequencing library, constructed with the last releases of pore chemistry (R9.4) and sequencing kit (SQK-LSK108), permitted the retrieval of the higher level of 1D read accuracy sufficient to characterize the microbial species present in each mock community analysed. Improvements in nanopore chemistry, such as minimizing base-calling errors and new library protocols able to produce rapid 1D libraries, will provide more reliable information in the near future. Such data will be useful for more comprehensive and faster specific detection of microbial species and strains in complex ecosystems. PMID:28605506

DNA migration mechanism analyses for applications in capillary and microchip electrophoresis

PubMed Central

Forster, Ryan E.; Hert, Daniel G.; Chiesl, Thomas N.; Fredlake, Christopher P.; Barron, Annelise E.

2009-01-01

In 2009, electrophoretically driven DNA separations in slab gels and capillaries have the sepia tones of an old-fashioned technology in the eyes of many, even while they remain ubiquitously used, fill a unique niche, and arguably have yet to reach their full potential. For comic relief, what is old becomes new again: agarose slab gel separations are used to prepare DNA samples for “next-gen” sequencing platforms (e.g., the Illumina and 454 machines)—dsDNA molecules within a certain size range are “cut out” of a gel and recovered for subsequent “massively parallel” pyrosequencing. In this review, we give a Barron lab perspective on how our comprehension of DNA migration mechanisms in electrophoresis has evolved, since the first reports of DNA separations by CE (∼1989) until now, 20 years later. Fused silica capillaries, and borosilicate glass and plastic microchips, quietly offer increasing capacities for fast (and even “ultra-fast”), efficient DNA separations. While the channel-by-channel scaling of both old and new electrophoresis platforms provides key flexibility, it requires each unique DNA sample to be prepared in its own micro- or nanovolume. This Achille's heel of electrophoresis technologies left an opening through which pooled-sample, next-gen DNA sequencing technologies rushed. We shall see, over time, whether sharpening understanding of transitions in DNA migration modes in crosslinked gels, nanogel solutions, and uncrosslinked polymer solutions will allow electrophoretic DNA analysis technologies to flower again. Microchannel electrophoresis, after a quiet period of metamorphosis, may emerge sleeker and more powerful, to claim its own important niche applications. PMID:19582705

Primer and platform effects on 16S rRNA tag sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tremblay, Julien; Singh, Kanwar; Fern, Alison

Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as wellmore » as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.« less

Primer and platform effects on 16S rRNA tag sequencing

DOE PAGES

Tremblay, Julien; Singh, Kanwar; Fern, Alison; ...

2015-08-04

Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as wellmore » as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.« less

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities.

PubMed

Deiner, Kristy; Bik, Holly M; Mächler, Elvira; Seymour, Mathew; Lacoursière-Roussel, Anaïs; Altermatt, Florian; Creer, Simon; Bista, Iliana; Lodge, David M; de Vere, Natasha; Pfrender, Michael E; Bernatchez, Louis

2017-11-01

The genomic revolution has fundamentally changed how we survey biodiversity on earth. High-throughput sequencing ("HTS") platforms now enable the rapid sequencing of DNA from diverse kinds of environmental samples (termed "environmental DNA" or "eDNA"). Coupling HTS with our ability to associate sequences from eDNA with a taxonomic name is called "eDNA metabarcoding" and offers a powerful molecular tool capable of noninvasively surveying species richness from many ecosystems. Here, we review the use of eDNA metabarcoding for surveying animal and plant richness, and the challenges in using eDNA approaches to estimate relative abundance. We highlight eDNA applications in freshwater, marine and terrestrial environments, and in this broad context, we distill what is known about the ability of different eDNA sample types to approximate richness in space and across time. We provide guiding questions for study design and discuss the eDNA metabarcoding workflow with a focus on primers and library preparation methods. We additionally discuss important criteria for consideration of bioinformatic filtering of data sets, with recommendations for increasing transparency. Finally, looking to the future, we discuss emerging applications of eDNA metabarcoding in ecology, conservation, invasion biology, biomonitoring, and how eDNA metabarcoding can empower citizen science and biodiversity education. © 2017 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

High-density fiber optic biosensor arrays

NASA Astrophysics Data System (ADS)

Epstein, Jason R.; Walt, David R.

2002-02-01

Novel approaches are required to coordinate the immense amounts of information derived from diverse genomes. This concept has influenced the expanded role of high-throughput DNA detection and analysis in the biological sciences. A high-density fiber optic DNA biosensor was developed consisting of oligonucleotide-functionalized, 3.1 mm diameter microspheres deposited into the etched wells on the distal face of a 500 micrometers imaging fiber bundle. Imaging fiber bundles containing thousands of optical fibers, each associated with a unique oligonucleotide probe sequence, were the foundation for an optically connected, individually addressable DNA detection platform. Different oligonucleotide-functionalized microspheres were combined in a stock solution, and randomly dispersed into the etched wells. Microsphere positions were registered from optical dyes incorporated onto the microspheres. The distribution process provided an inherent redundancy that increases the signal-to-noise ratio as the square root of the number of sensors examined. The representative amount of each probe-type in the array was dependent on their initial stock solution concentration, and as other sequences of interest arise, new microsphere elements can be added to arrays without altering the existing detection capabilities. The oligonucleotide probe sequences hybridize to fluorescently-labeled, complementary DNA target solutions. Fiber optic DNA microarray research has included DNA-protein interaction profiles, microbial strain differentiation, non-labeled target interrogation with molecular beacons, and single cell-based assays. This biosensor array is proficient in DNA detection linked to specific disease states, single nucleotide polymorphism (SNP's) discrimination, and gene expression analysis. This array platform permits multiple detection formats, provides smaller feature sizes, and enables sensor design flexibility. High-density fiber optic microarray biosensors provide a fast, reversible format with the detection limit of a few hundred molecules.

Detection of BRAF mutations from solid tumors using Tumorplex™ technology

PubMed Central

Yo, Jacob; Hay, Katie S.L.; Vinayagamoorthy, Dilanthi; Maryanski, Danielle; Carter, Mark; Wiegel, Joseph; Vinayagamoorthy, Thuraiayah

2015-01-01

Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. • Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal. • Tumorplex™ provides a high level of sensitivity. • Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA. PMID:26258049

Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean.

PubMed

Manzanares-Palenzuela, C Lorena; de-Los-Santos-Álvarez, Noemí; Lobo-Castañón, María Jesús; López-Ruiz, Beatriz

2015-06-15

Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2-250 pM for both targets, with LOD values of 650 fM (160 amol) and 190 fM (50 amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods. Copyright © 2015 Elsevier B.V. All rights reserved.

Sputnik: a database platform for comparative plant genomics.

PubMed

Rudd, Stephen; Mewes, Hans-Werner; Mayer, Klaus F X

2003-01-01

Two million plant ESTs, from 20 different plant species, and totalling more than one 1000 Mbp of DNA sequence, represents a formidable transcriptomic resource. Sputnik uses the potential of this sequence resource to fill some of the information gap in the un-sequenced plant genomes and to serve as the foundation for in silicio comparative plant genomics. The complexity of the individual EST collections has been reduced using optimised EST clustering techniques. Annotation of cluster sequences is performed by exploiting and transferring information from the comprehensive knowledgebase already produced for the completed model plant genome (Arabidopsis thaliana) and by performing additional state of-the-art sequence analyses relevant to today's plant biologist. Functional predictions, comparative analyses and associative annotations for 500 000 plant EST derived peptides make Sputnik (http://mips.gsf.de/proj/sputnik/) a valid platform for contemporary plant genomics.

Sputnik: a database platform for comparative plant genomics

PubMed Central

Rudd, Stephen; Mewes, Hans-Werner; Mayer, Klaus F.X.

2003-01-01

Two million plant ESTs, from 20 different plant species, and totalling more than one 1000 Mbp of DNA sequence, represents a formidable transcriptomic resource. Sputnik uses the potential of this sequence resource to fill some of the information gap in the un-sequenced plant genomes and to serve as the foundation for in silicio comparative plant genomics. The complexity of the individual EST collections has been reduced using optimised EST clustering techniques. Annotation of cluster sequences is performed by exploiting and transferring information from the comprehensive knowledgebase already produced for the completed model plant genome (Arabidopsis thaliana) and by performing additional state of-the-art sequence analyses relevant to today's plant biologist. Functional predictions, comparative analyses and associative annotations for 500 000 plant EST derived peptides make Sputnik (http://mips.gsf.de/proj/sputnik/) a valid platform for contemporary plant genomics. PMID:12519965

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes.

PubMed

Fredlake, Christopher P; Hert, Daniel G; Kan, Cheuk-Wai; Chiesl, Thomas N; Root, Brian E; Forster, Ryan E; Barron, Annelise E

2008-01-15

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require approximately 70 min to deliver approximately 650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered "hybrid" mechanism of DNA electromigration, in which DNA molecules alternate rapidly between repeating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs.

Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes

PubMed Central

Fredlake, Christopher P.; Hert, Daniel G.; Kan, Cheuk-Wai; Chiesl, Thomas N.; Root, Brian E.; Forster, Ryan E.; Barron, Annelise E.

2008-01-01

To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require ≈70 min to deliver ≈650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered “hybrid” mechanism of DNA electromigration, in which DNA molecules alternate rapidly between reptating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs. PMID:18184818

Complete Genome Sequence of ER2796, a DNA Methyltransferase-Deficient Strain of Escherichia coli K-12.

PubMed

Anton, Brian P; Mongodin, Emmanuel F; Agrawal, Sonia; Fomenkov, Alexey; Byrd, Devon R; Roberts, Richard J; Raleigh, Elisabeth A

2015-01-01

We report the complete sequence of ER2796, a laboratory strain of Escherichia coli K-12 that is completely defective in DNA methylation. Because of its lack of any native methylation, it is extremely useful as a host into which heterologous DNA methyltransferase genes can be cloned and the recognition sequences of their products deduced by Pacific Biosciences Single-Molecule Real Time (SMRT) sequencing. The genome was itself sequenced from a long-insert library using the SMRT platform, resulting in a single closed contig devoid of methylated bases. Comparison with K-12 MG1655, the first E. coli K-12 strain to be sequenced, shows an essentially co-linear relationship with no major rearrangements despite many generations of laboratory manipulation. The comparison revealed a total of 41 insertions and deletions, and 228 single base pair substitutions. In addition, the long-read approach facilitated the surprising discovery of four gene conversion events, three involving rRNA operons and one between two cryptic prophages. Such events thus contribute both to genomic homogenization and to bacteriophage diversification. As one of relatively few laboratory strains of E. coli to be sequenced, the genome also reveals the sequence changes underlying a number of classical mutant alleles including those affecting the various native DNA methylation systems.

Complete Genome Sequence of ER2796, a DNA Methyltransferase-Deficient Strain of Escherichia coli K-12

PubMed Central

Anton, Brian P.; Mongodin, Emmanuel F.; Agrawal, Sonia; Fomenkov, Alexey; Byrd, Devon R.; Roberts, Richard J.; Raleigh, Elisabeth A.

2015-01-01

We report the complete sequence of ER2796, a laboratory strain of Escherichia coli K-12 that is completely defective in DNA methylation. Because of its lack of any native methylation, it is extremely useful as a host into which heterologous DNA methyltransferase genes can be cloned and the recognition sequences of their products deduced by Pacific Biosciences Single-Molecule Real Time (SMRT) sequencing. The genome was itself sequenced from a long-insert library using the SMRT platform, resulting in a single closed contig devoid of methylated bases. Comparison with K-12 MG1655, the first E. coli K-12 strain to be sequenced, shows an essentially co-linear relationship with no major rearrangements despite many generations of laboratory manipulation. The comparison revealed a total of 41 insertions and deletions, and 228 single base pair substitutions. In addition, the long-read approach facilitated the surprising discovery of four gene conversion events, three involving rRNA operons and one between two cryptic prophages. Such events thus contribute both to genomic homogenization and to bacteriophage diversification. As one of relatively few laboratory strains of E. coli to be sequenced, the genome also reveals the sequence changes underlying a number of classical mutant alleles including those affecting the various native DNA methylation systems. PMID:26010885

MIG-seq: an effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform

PubMed Central

Suyama, Yoshihisa; Matsuki, Yu

2015-01-01

Restriction-enzyme (RE)-based next-generation sequencing methods have revolutionized marker-assisted genetic studies; however, the use of REs has limited their widespread adoption, especially in field samples with low-quality DNA and/or small quantities of DNA. Here, we developed a PCR-based procedure to construct reduced representation libraries without RE digestion steps, representing de novo single-nucleotide polymorphism discovery, and its genotyping using next-generation sequencing. Using multiplexed inter-simple sequence repeat (ISSR) primers, thousands of genome-wide regions were amplified effectively from a wide variety of genomes, without prior genetic information. We demonstrated: 1) Mendelian gametic segregation of the discovered variants; 2) reproducibility of genotyping by checking its applicability for individual identification; and 3) applicability in a wide variety of species by checking standard population genetic analysis. This approach, called multiplexed ISSR genotyping by sequencing, should be applicable to many marker-assisted genetic studies with a wide range of DNA qualities and quantities. PMID:26593239

Impedimetric DNA biosensor based on a nanoporous alumina membrane for the detection of the specific oligonucleotide sequence of dengue virus.

PubMed

Deng, Jiajia; Toh, Chee-Seng

2013-06-17

A novel and integrated membrane sensing platform for DNA detection is developed based on an anodic aluminum oxide (AAO) membrane. Platinum electrodes (~50-100 nm thick) are coated directly on both sides of the alumina membrane to eliminate the solution resistance outside the nanopores. The electrochemical impedance technique is employed to monitor the impedance changes within the nanopores upon DNA binding. Pore resistance (Rp) linearly increases in response towards the increasing concentration of the target DNA in the range of 1 × 10⁻¹² to 1 × 10⁻⁶ M. Moreover, the biosensor selectively differentiates the complementary sequence from single base mismatched (MM-1) strands and non-complementary strands. This study reveals a simple, selective and sensitive method to fabricate a label-free DNA biosensor.

An enhanced sensing platform for ultrasensitive impedimetric detection of target genes based on ordered FePt nanoparticles decorated carbon nanotubes.

PubMed

Zhang, Wei; Zong, Peisong; Zheng, Xiuwen; Wang, Libin

2013-04-15

We demonstrate a novel high-performance DNA hybridization biosensor with a carbon nanotubes (CNTs)-based nanocomposite membrane as the enhanced sensing platform. The platform was constructed by homogenously distributing ordered FePt nanoparticles (NPs) onto the CNTs matrix. The surface structure and electrochemical performance of the FePt/CNTs nanocomposite membrane were systematically investigated. Such a nanostructured composite membrane platform could combine with the advantages of FePt NPs and CNTs, greatly facilitate the electron-transfer process and the sensing behavior for DNA detection, leading to excellent sensitivity and selectivity. The complementary target genes from acute promyelocytic leukemia could be quantified in a wide range of 1.0×10⁻¹² mol/L to 1.0×10⁻⁶ mol/L using electrochemical impedance spectroscopy, and the detection limit was 2.1×10⁻¹³ mol/L under the optimal conditions. In addition, the DNA electrochemical biosensor was highly selective to discriminate single-base or double-base mismatched sequences. Copyright © 2012 Elsevier B.V. All rights reserved.

Design and characterization of a nanopore-coupled polymerase for single-molecule DNA sequencing by synthesis on an electrode array

PubMed Central

Stranges, P. Benjamin; Palla, Mirkó; Kalachikov, Sergey; Nivala, Jeff; Dorwart, Michael; Trans, Andrew; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Tao, Chuanjuan; Morozova, Irina; Li, Zengmin; Shi, Shundi; Aberra, Aman; Arnold, Cleoma; Yang, Alexander; Aguirre, Anne; Harada, Eric T.; Korenblum, Daniel; Pollard, James; Bhat, Ashwini; Gremyachinskiy, Dmitriy; Bibillo, Arek; Chen, Roger; Davis, Randy; Russo, James J.; Fuller, Carl W.; Roever, Stefan; Ju, Jingyue; Church, George M.

2016-01-01

Scalable, high-throughput DNA sequencing is a prerequisite for precision medicine and biomedical research. Recently, we presented a nanopore-based sequencing-by-synthesis (Nanopore-SBS) approach, which used a set of nucleotides with polymer tags that allow discrimination of the nucleotides in a biological nanopore. Here, we designed and covalently coupled a DNA polymerase to an α-hemolysin (αHL) heptamer using the SpyCatcher/SpyTag conjugation approach. These porin–polymerase conjugates were inserted into lipid bilayers on a complementary metal oxide semiconductor (CMOS)-based electrode array for high-throughput electrical recording of DNA synthesis. The designed nanopore construct successfully detected the capture of tagged nucleotides complementary to a DNA base on a provided template. We measured over 200 tagged-nucleotide signals for each of the four bases and developed a classification method to uniquely distinguish them from each other and background signals. The probability of falsely identifying a background event as a true capture event was less than 1.2%. In the presence of all four tagged nucleotides, we observed sequential additions in real time during polymerase-catalyzed DNA synthesis. Single-polymerase coupling to a nanopore, in combination with the Nanopore-SBS approach, can provide the foundation for a low-cost, single-molecule, electronic DNA-sequencing platform. PMID:27729524

Methodologic European external quality assurance for DNA sequencing: the EQUALseq program.

PubMed

Ahmad-Nejad, Parviz; Dorn-Beineke, Alexandra; Pfeiffer, Ulrike; Brade, Joachim; Geilenkeuser, Wolf-Jochen; Ramsden, Simon; Pazzagli, Mario; Neumaier, Michael

2006-04-01

DNA sequencing is a key technique in molecular diagnostics, but to date no comprehensive methodologic external quality assessment (EQA) programs have been instituted. Between 2003 and 2005, the European Union funded, as specific support actions, the EQUAL initiative to develop methodologic EQA schemes for genotyping (EQUALqual), quantitative PCR (EQUALquant), and sequencing (EQUALseq). Here we report on the results of the EQUALseq program. The participating laboratories received a 4-sample set comprising 2 DNA plasmids, a PCR product, and a finished sequencing reaction to be analyzed. Data and information from detailed questionnaires were uploaded online and evaluated by use of a scoring system for technical skills and proficiency of data interpretation. Sixty laboratories from 21 European countries registered, and 43 participants (72%) returned data and samples. Capillary electrophoresis was the predominant platform (n = 39; 91%). The median contiguous correct sequence stretch was 527 nucleotides with considerable variation in quality of both primary data and data evaluation. The association between laboratory performance and the number of sequencing assays/year was statistically significant (P <0.05). Interestingly, more than 30% of participants neither added comments to their data nor made efforts to identify the gene sequences or mutational positions. Considerable variations exist even in a highly standardized methodology such as DNA sequencing. Methodologic EQAs are appropriate tools to uncover strengths and weaknesses in both technique and proficiency, and our results emphasize the need for mandatory EQAs. The results of EQUALseq should help improve the overall quality of molecular genetics findings obtained by DNA sequencing.

An integrated pipeline for next generation sequencing and annotation of the complete mitochondrial genome of the giant intestinal fluke, Fasciolopsis buski (Lankester, 1857) Looss, 1899

PubMed Central

Biswal, Devendra Kumar; Ghatani, Sudeep; Shylla, Jollin A.; Sahu, Ranjana; Mullapudi, Nandita

2013-01-01

Helminths include both parasitic nematodes (roundworms) and platyhelminths (trematode and cestode flatworms) that are abundant, and are of clinical importance. The genetic characterization of parasitic flatworms using advanced molecular tools is central to the diagnosis and control of infections. Although the nuclear genome houses suitable genetic markers (e.g., in ribosomal (r) DNA) for species identification and molecular characterization, the mitochondrial (mt) genome consistently provides a rich source of novel markers for informative systematics and epidemiological studies. In the last decade, there have been some important advances in mtDNA genomics of helminths, especially lung flukes, liver flukes and intestinal flukes. Fasciolopsis buski, often called the giant intestinal fluke, is one of the largest digenean trematodes infecting humans and found primarily in Asia, in particular the Indian subcontinent. Next-generation sequencing (NGS) technologies now provide opportunities for high throughput sequencing, assembly and annotation within a short span of time. Herein, we describe a high-throughput sequencing and bioinformatics pipeline for mt genomics for F. buski that emphasizes the utility of short read NGS platforms such as Ion Torrent and Illumina in successfully sequencing and assembling the mt genome using innovative approaches for PCR primer design as well as assembly. We took advantage of our NGS whole genome sequence data (unpublished so far) for F. buski and its comparison with available data for the Fasciola hepatica mtDNA as the reference genome for design of precise and specific primers for amplification of mt genome sequences from F. buski. A long-range PCR was carried out to create an NGS library enriched in mt DNA sequences. Two different NGS platforms were employed for complete sequencing, assembly and annotation of the F. buski mt genome. The complete mt genome sequences of the intestinal fluke comprise 14,118 bp and is thus the shortest trematode mitochondrial genome sequenced to date. The noncoding control regions are separated into two parts by the tRNA-Gly gene and don’t contain either tandem repeats or secondary structures, which are typical for trematode control regions. The gene content and arrangement are identical to that of F. hepatica. The F. buski mtDNA genome has a close resemblance with F. hepatica and has a similar gene order tallying with that of other trematodes. The mtDNA for the intestinal fluke is reported herein for the first time by our group that would help investigate Fasciolidae taxonomy and systematics with the aid of mtDNA NGS data. More so, it would serve as a resource for comparative mitochondrial genomics and systematic studies of trematode parasites. PMID:24255820

Separation and parallel sequencing of the genomes and transcriptomes of single cells using G&T-seq.

PubMed

Macaulay, Iain C; Teng, Mabel J; Haerty, Wilfried; Kumar, Parveen; Ponting, Chris P; Voet, Thierry

2016-11-01

Parallel sequencing of a single cell's genome and transcriptome provides a powerful tool for dissecting genetic variation and its relationship with gene expression. Here we present a detailed protocol for G&T-seq, a method for separation and parallel sequencing of genomic DNA and full-length polyA(+) mRNA from single cells. We provide step-by-step instructions for the isolation and lysis of single cells; the physical separation of polyA(+) mRNA from genomic DNA using a modified oligo-dT bead capture and the respective whole-transcriptome and whole-genome amplifications; and library preparation and sequence analyses of these amplification products. The method allows the detection of thousands of transcripts in parallel with the genetic variants captured by the DNA-seq data from the same single cell. G&T-seq differs from other currently available methods for parallel DNA and RNA sequencing from single cells, as it involves physical separation of the DNA and RNA and does not require bespoke microfluidics platforms. The process can be implemented manually or through automation. When performed manually, paired genome and transcriptome sequencing libraries from eight single cells can be produced in ∼3 d by researchers experienced in molecular laboratory work. For users with experience in the programming and operation of liquid-handling robots, paired DNA and RNA libraries from 96 single cells can be produced in the same time frame. Sequence analysis and integration of single-cell G&T-seq DNA and RNA data requires a high level of bioinformatics expertise and familiarity with a wide range of informatics tools.

Methodology challenges in studying human gut microbiota - effects of collection, storage, DNA extraction and next generation sequencing technologies.

PubMed

Panek, Marina; Čipčić Paljetak, Hana; Barešić, Anja; Perić, Mihaela; Matijašić, Mario; Lojkić, Ivana; Vranešić Bender, Darija; Krznarić, Željko; Verbanac, Donatella

2018-03-23

The information on microbiota composition in the human gastrointestinal tract predominantly originates from the analyses of human faeces by application of next generation sequencing (NGS). However, the detected composition of the faecal bacterial community can be affected by various factors including experimental design and procedures. This study evaluated the performance of different protocols for collection and storage of faecal samples (native and OMNIgene.GUT system) and bacterial DNA extraction (MP Biomedicals, QIAGEN and MO BIO kits), using two NGS platforms for 16S rRNA gene sequencing (Ilumina MiSeq and Ion Torrent PGM). OMNIgene.GUT proved as a reliable and convenient system for collection and storage of faecal samples although favouring Sutterella genus. MP provided superior DNA yield and quality, MO BIO depleted Gram positive organisms while using QIAGEN with OMNIgene.GUT resulted in greatest variability compared to other two kits. MiSeq and IT platforms in their supplier recommended setups provided comparable reproducibility of donor faecal microbiota. The differences included higher diversity observed with MiSeq and increased capacity of MiSeq to detect Akkermansia muciniphila, [Odoribacteraceae], Erysipelotrichaceae and Ruminococcaceae (primarily Faecalibacterium prausnitzii). The results of our study could assist the investigators using NGS technologies to make informed decisions on appropriate tools for their experimental pipelines.

The skin microbiome in psoriatic arthritis: methodology development and pilot data.

PubMed

Castelino, Madhura; Eyre, Stephen; Moat, John; Fox, Graeme; Martin, Paul; Ijaz, Umer; Quince, Christopher; Ho, Pauline; Upton, Mathew; Barton, Anne

2015-02-26

Skin microbiota are likely to be important in the development of conditions such as psoriatic arthritis. Profiling the bacterial community in the psosriatic plaques will contribute to our understanding of the role of the skin microbiome in these conditions. The aim of this work was to determine the optimum study design for work on the skin microbiome with use of the MiSeq platform. The objectives were to compare data generated from two platforms for two primer pairs in a low density mock bacterial community. DNA was obtained from two low density mock communities of 11 diverse bacterial strains (with and without human DNA supplementation) and from swabs taken from the skin of four healthy volunteers. The DNA was amplified with primer pairs covering hypervariable regions of the 16S rRNA gene: primers 63F and 519R (V1-V3), and 347F and 803R (V3-V4). The resultant libraries were indexed for the MiSeq and Roche454 platforms and sequenced. Both datasets were de-noised, cleaned of chimeras, and analysed by use of QIIME software (version 1.8.0). No significant difference in the diversity indices at the phylum and the genus level between the platforms was seen. Comparison of the diversity indices for the mock community data for the two primer pairs demonstrated that the V3-V4 hypervariable region had significantly better capture of bacterial diversity than did the V1-V3 region. Amplification with the same primer pairs showed strong concordance within each platform (98·9-99·8%), with negligible effect of spiked human DNA contamination. Comparison at the family level classification between samples processed on the MiSeq and Roche454 platforms using the V3-V4 hypervariable region also showed a high level of concordance (87%), although less so for the V1-V3 primers (10%). The pilot data from healthy volunteers were similar. Results obtained from the V3-V4 16S rRNA hypervariable region, sequencing on the MiSeq and Roche454 platforms, were concordant between replicates, and between each other. These findings suggest that the MiSeq platform, and these primers, is a comparable method for determining skin microbiota to the widely used Roche454 methodology. NIHR Manchester Musculoskeletal Biomedical Research Unit. Copyright © 2015 Elsevier Ltd. All rights reserved.

Clinical Utility of Circulating Tumor DNA for Molecular Assessment and Precision Medicine in Pancreatic Cancer.

PubMed

Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Kato, Mamoru; Shibata, Tatsuhiro; Yachida, Shinichi

2016-01-01

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect molecular characteristics of tumors, supporting the concept of "liquid biopsy".We determined the mutational status of KRAS in plasma cfDNA using multiplex droplet digital PCR in 259 patients with PDAC, retrospectively. Furthermore, we constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA in 48 patients who had ≥1 % mutant allele frequencies of KRAS in plasma cfDNA.Droplet digital PCR detected KRAS mutations in plasma cfDNA in 63 of 107 (58.9 %) patients with inoperable tumors. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2 %) examined by cfDNA sequencing.Our two-step approach with plasma cfDNA, combining droplet digital PCR and targeted deep sequencing, is a feasible clinical approach. Assessment of mutations in plasma cfDNA may provide a new diagnostic tool, assisting decisions for optimal therapeutic strategies for PDAC patients.

Comparison of Ion Personal Genome Machine Platforms for the Detection of Variants in BRCA1 and BRCA2.

PubMed

Hwang, Sang Mee; Lee, Ki Chan; Lee, Min Seob; Park, Kyoung Un

2018-01-01

Transition to next generation sequencing (NGS) for BRCA1 / BRCA2 analysis in clinical laboratories is ongoing but different platforms and/or data analysis pipelines give different results resulting in difficulties in implementation. We have evaluated the Ion Personal Genome Machine (PGM) Platforms (Ion PGM, Ion PGM Dx, Thermo Fisher Scientific) for the analysis of BRCA1 /2. The results of Ion PGM with OTG-snpcaller, a pipeline based on Torrent mapping alignment program and Genome Analysis Toolkit, from 75 clinical samples and 14 reference DNA samples were compared with Sanger sequencing for BRCA1 / BRCA2 . Ten clinical samples and 14 reference DNA samples were additionally sequenced by Ion PGM Dx with Torrent Suite. Fifty types of variants including 18 pathogenic or variants of unknown significance were identified from 75 clinical samples and known variants of the reference samples were confirmed by Sanger sequencing and/or NGS. One false-negative results were present for Ion PGM/OTG-snpcaller for an indel variant misidentified as a single nucleotide variant. However, eight discordant results were present for Ion PGM Dx/Torrent Suite with both false-positive and -negative results. A 40-bp deletion, a 4-bp deletion and a 1-bp deletion variant was not called and a false-positive deletion was identified. Four other variants were misidentified as another variant. Ion PGM/OTG-snpcaller showed acceptable performance with good concordance with Sanger sequencing. However, Ion PGM Dx/Torrent Suite showed many discrepant results not suitable for use in a clinical laboratory, requiring further optimization of the data analysis for calling variants.

High-density, microsphere-based fiber optic DNA microarrays.

PubMed

Epstein, Jason R; Leung, Amy P K; Lee, Kyong Hoon; Walt, David R

2003-05-01

A high-density fiber optic DNA microarray has been developed consisting of oligonucleotide-functionalized, 3.1-microm-diameter microspheres randomly distributed on the etched face of an imaging fiber bundle. The fiber bundles are comprised of 6000-50000 fused optical fibers and each fiber terminates with an etched well. The microwell array is capable of housing complementary-sized microspheres, each containing thousands of copies of a unique oligonucleotide probe sequence. The array fabrication process results in random microsphere placement. Determining the position of microspheres in the random array requires an optical encoding scheme. This array platform provides many advantages over other array formats. The microsphere-stock suspension concentration added to the etched fiber can be controlled to provide inherent sensor redundancy. Examining identical microspheres has a beneficial effect on the signal-to-noise ratio. As other sequences of interest are discovered, new microsphere sensing elements can be added to existing microsphere pools and new arrays can be fabricated incorporating the new sequences without altering the existing detection capabilities. These microarrays contain the smallest feature sizes (3 microm) of any DNA array, allowing interrogation of extremely small sample volumes. Reducing the feature size results in higher local target molecule concentrations, creating rapid and highly sensitive assays. The microsphere array platform is also flexible in its applications; research has included DNA-protein interaction profiles, microbial strain differentiation, and non-labeled target interrogation with molecular beacons. Fiber optic microsphere-based DNA microarrays have a simple fabrication protocol enabling their expansion into other applications, such as single cell-based assays.

Historical Perspective, Development and Applications of Next-Generation Sequencing in Plant Virology

PubMed Central

Barba, Marina; Czosnek, Henryk; Hadidi, Ahmed

2014-01-01

Next-generation high throughput sequencing technologies became available at the onset of the 21st century. They provide a highly efficient, rapid, and low cost DNA sequencing platform beyond the reach of the standard and traditional DNA sequencing technologies developed in the late 1970s. They are continually improved to become faster, more efficient and cheaper. They have been used in many fields of biology since 2004. In 2009, next-generation sequencing (NGS) technologies began to be applied to several areas of plant virology including virus/viroid genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. Identification and characterization of known and unknown viruses and/or viroids in infected plants are currently among the most successful applications of these technologies. It is expected that NGS will play very significant roles in many research and non-research areas of plant virology. PMID:24399207

Protein-binding aptamer assisted signal amplification for the detection of influenza A (H1N1) DNA sequences based on quantum dot fluorescence polarization analysis.

PubMed

Zhang, Juanni; Tian, Jianniao; He, Yanlong; Chen, Sheng; Jiang, Yixuan; Zhao, Yanchun; Zhao, Shulin

2013-09-07

We report a fluorescence polarization platform for H1N1 detection based on the construction of a DNA functional QD fluorescence polarization probe and a bi-functional protein binding aptamer (Apt-DNA). The assay has a linear range from 10 nM to 100 nM with a detection limit of 3.45 nM and is selective over the mismatched bases.

Comparison and evaluation of two exome capture kits and sequencing platforms for variant calling.

PubMed

Zhang, Guoqiang; Wang, Jianfeng; Yang, Jin; Li, Wenjie; Deng, Yutian; Li, Jing; Huang, Jun; Hu, Songnian; Zhang, Bing

2015-08-05

To promote the clinical application of next-generation sequencing, it is important to obtain accurate and consistent variants of target genomic regions at low cost. Ion Proton, the latest updated semiconductor-based sequencing instrument from Life Technologies, is designed to provide investigators with an inexpensive platform for human whole exome sequencing that achieves a rapid turnaround time. However, few studies have comprehensively compared and evaluated the accuracy of variant calling between Ion Proton and Illumina sequencing platforms such as HiSeq 2000, which is the most popular sequencing platform for the human genome. The Ion Proton sequencer combined with the Ion TargetSeq Exome Enrichment Kit together make up TargetSeq-Proton, whereas SureSelect-Hiseq is based on the Agilent SureSelect Human All Exon v4 Kit and the HiSeq 2000 sequencer. Here, we sequenced exonic DNA from four human blood samples using both TargetSeq-Proton and SureSelect-HiSeq. We then called variants in the exonic regions that overlapped between the two exome capture kits (33.6 Mb). The rates of shared variant loci called by two sequencing platforms were from 68.0 to 75.3% in four samples, whereas the concordance of co-detected variant loci reached 99%. Sanger sequencing validation revealed that the validated rate of concordant single nucleotide polymorphisms (SNPs) (91.5%) was higher than the SNPs specific to TargetSeq-Proton (60.0%) or specific to SureSelect-HiSeq (88.3%). With regard to 1-bp small insertions and deletions (InDels), the Sanger sequencing validated rates of concordant variants (100.0%) and SureSelect-HiSeq-specific (89.6%) were higher than those of TargetSeq-Proton-specific (15.8%). In the sequencing of exonic regions, a combination of using of two sequencing strategies (SureSelect-HiSeq and TargetSeq-Proton) increased the variant calling specificity for concordant variant loci and the sensitivity for variant loci called by any one platform. However, for the sequencing of platform-specific variants, the accuracy of variant calling by HiSeq 2000 was higher than that of Ion Proton, specifically for the InDel detection. Moreover, the variant calling software also influences the detection of SNPs and, specifically, InDels in Ion Proton exome sequencing.

A Fast Solution to NGS Library Prep with Low Nanogram DNA Input

PubMed Central

Liu, Pingfang; Lohman, Gregory J.S.; Cantor, Eric; Langhorst, Bradley W.; Yigit, Erbay; Apone, Lynne M.; Munafo, Daniela B.; Stewart, Fiona J.; Evans, Thomas C.; Nichols, Nicole; Dimalanta, Eileen T.; Davis, Theodore B.; Sumner, Christine

2013-01-01

Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.

Biomimetic nanochannels based biosensor for ultrasensitive and label-free detection of nucleic acids.

PubMed

Sun, Zhongyue; Liao, Tangbin; Zhang, Yulin; Shu, Jing; Zhang, Hong; Zhang, Guo-Jun

2016-12-15

A very simple sensing device based on biomimetic nanochannels has been developed for label-free, ultrasensitive and highly sequence-specific detection of DNA. Probe DNA was modified on the inner wall of the nanochannel surface by layer-by-layer (LBL) assembly. After probe DNA immobilization, DNA detection was realized by monitoring the rectified ion current when hybridization occurred. Due to three dimensional (3D) nanoscale environment of the nanochannel, this special geometry dramatically increased the surface area of the nanochannel for immobilization of probe molecules on the inner-surface and enlarged contact area between probes and target-molecules. Thus, the unique sensor reached a reliable detection limit of 10 fM for target DNA. In addition, this DNA sensor could discriminate complementary DNA (c-DNA) from non-complementary DNA (nc-DNA), two-base mismatched DNA (2bm-DNA) and one-base mismatched DNA (1bm-DNA) with high specificity. Moreover, the nanochannel-based biosensor was also able to detect target DNA even in an interfering environment and serum samples. This approach will provide a novel biosensing platform for detection and discrimination of disease-related molecular targets and unknown sequence DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

Diversity arrays technology: a generic genome profiling technology on open platforms.

PubMed

Kilian, Andrzej; Wenzl, Peter; Huttner, Eric; Carling, Jason; Xia, Ling; Blois, Hélène; Caig, Vanessa; Heller-Uszynska, Katarzyna; Jaccoud, Damian; Hopper, Colleen; Aschenbrenner-Kilian, Malgorzata; Evers, Margaret; Peng, Kaiman; Cayla, Cyril; Hok, Puthick; Uszynski, Grzegorz

2012-01-01

In the last 20 years, we have observed an exponential growth of the DNA sequence data and simular increase in the volume of DNA polymorphism data generated by numerous molecular marker technologies. Most of the investment, and therefore progress, concentrated on human genome and genomes of selected model species. Diversity Arrays Technology (DArT), developed over a decade ago, was among the first "democratizing" genotyping technologies, as its performance was primarily driven by the level of DNA sequence variation in the species rather than by the level of financial investment. DArT also proved more robust to genome size and ploidy-level differences among approximately 60 organisms for which DArT was developed to date compared to other high-throughput genotyping technologies. The success of DArT in a number of organisms, including a wide range of "orphan crops," can be attributed to the simplicity of underlying concepts: DArT combines genome complexity reduction methods enriching for genic regions with a highly parallel assay readout on a number of "open-access" microarray platforms. The quantitative nature of the assay enabled a number of applications in which allelic frequencies can be estimated from DArT arrays. A typical DArT assay tests for polymorphism tens of thousands of genomic loci with the final number of markers reported (hundreds to thousands) reflecting the level of DNA sequence variation in the tested loci. Detailed DArT methods, protocols, and a range of their application examples as well as DArT's evolution path are presented.

Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer.

PubMed

Takai, Erina; Totoki, Yasushi; Nakamura, Hiromi; Morizane, Chigusa; Nara, Satoshi; Hama, Natsuko; Suzuki, Masami; Furukawa, Eisaku; Kato, Mamoru; Hayashi, Hideyuki; Kohno, Takashi; Ueno, Hideki; Shimada, Kazuaki; Okusaka, Takuji; Nakagama, Hitoshi; Shibata, Tatsuhiro; Yachida, Shinichi

2015-12-16

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. The genomic landscape of the PDAC genome features four frequently mutated genes (KRAS, CDKN2A, TP53, and SMAD4) and dozens of candidate driver genes altered at low frequency, including potential clinical targets. Circulating cell-free DNA (cfDNA) is a promising resource to detect and monitor molecular characteristics of tumors. In the present study, we determined the mutational status of KRAS in plasma cfDNA using multiplex picoliter-droplet digital PCR in 259 patients with PDAC. We constructed a novel modified SureSelect-KAPA-Illumina platform and an original panel of 60 genes. We then performed targeted deep sequencing of cfDNA and matched germline DNA samples in 48 patients who had ≥1% mutant allele frequencies of KRAS in plasma cfDNA. Importantly, potentially targetable somatic mutations were identified in 14 of 48 patients (29.2%) examined by targeted deep sequencing of cfDNA. We also analyzed somatic copy number alterations based on the targeted sequencing data using our in-house algorithm, and potentially targetable amplifications were detected. Assessment of mutations and copy number alterations in plasma cfDNA may provide a prognostic and diagnostic tool to assist decisions regarding optimal therapeutic strategies for PDAC patients.

Inhibition of HMGA2 binding to DNA by netropsin

PubMed Central

Miao, Yi; Cui, Tengjiao; Leng, Fenfei; Wilson, W. David

2008-01-01

The design of small synthetic molecules that can be used to affect gene expression is an area of active interest for development of agents in therapeutic and biotechnology applications. Many compounds that target the minor groove in AT sequences in DNA are well characterized and are promising reagents for use as modulators of protein-DNA complexes. The mammalian high mobility group transcriptional factor, HMGA2, also targets the DNA minor groove and plays critical roles in disease processes from cancer to obesity. Biosensor-surface plasmon resonance methods were used to monitor HMGA2 binding to target sites on immobilized DNA and a competition assay for inhibition of the HMGA2-DNA complex was designed. HMGA2 binds strongly to the DNA through AT hook domains with KD values of 20 - 30 nM depending on the DNA sequence. The well-characterized minor groove binder, netropsin, was used to develop and test the assay. The compound has two binding sites in the protein-DNA interaction sequence and this provides an advantage for inhibition. An equation for analysis of results when the inhibitor has two binding sites in the biopolymer recognition surface is presented with the results. The assay provides a platform for discovery of HMGA2 inhibitors. PMID:18023407

Alignment of high-throughput sequencing data inside in-memory databases.

PubMed

Firnkorn, Daniel; Knaup-Gregori, Petra; Lorenzo Bermejo, Justo; Ganzinger, Matthias

2014-01-01

In times of high-throughput DNA sequencing techniques, performance-capable analysis of DNA sequences is of high importance. Computer supported DNA analysis is still an intensive time-consuming task. In this paper we explore the potential of a new In-Memory database technology by using SAP's High Performance Analytic Appliance (HANA). We focus on read alignment as one of the first steps in DNA sequence analysis. In particular, we examined the widely used Burrows-Wheeler Aligner (BWA) and implemented stored procedures in both, HANA and the free database system MySQL, to compare execution time and memory management. To ensure that the results are comparable, MySQL has been running in memory as well, utilizing its integrated memory engine for database table creation. We implemented stored procedures, containing exact and inexact searching of DNA reads within the reference genome GRCh37. Due to technical restrictions in SAP HANA concerning recursion, the inexact matching problem could not be implemented on this platform. Hence, performance analysis between HANA and MySQL was made by comparing the execution time of the exact search procedures. Here, HANA was approximately 27 times faster than MySQL which means, that there is a high potential within the new In-Memory concepts, leading to further developments of DNA analysis procedures in the future.

Rapid, Affordable, and Point-of-Care Water Monitoring Via a Microfluidic DNA Sensor and a Mobile Interface for Global Health

PubMed Central

Ghanbari, Sarah; Ravikumar, Anusha; Seubert, John; Figueira, Silvia

2013-01-01

Contaminated water is a serious concern in many developing countries with severe health consequences particularly for children. Current methods for monitoring waterborne pathogens are often time consuming, expensive, and labor intensive, making them not suitable for these regions. Electrochemical detection in a microfluidic platform offers many advantages such as portability, minimal use of instrumentation, and easy integration with electronics. In many parts of the world, however, the required equipment for pathogen detection through electrochemical sensors is either not available or insufficiently portable, and operators may not be trained to use these sensors and interpret results, ultimately preventing its wide adoption. Counterintuitively, these same regions often have an extensive mobile phone infrastructure, suggesting the possibility of integrating electrochemical detection of bacterial pathogens with a mobile platform. Toward a solution to water quality interventions, we demonstrate a microfluidic electrochemical sensor combined with a mobile interface that detects the sequences from bacterial pathogens, suitable for rapid, affordable, and point-of-care water monitoring. We employ the transduction of DNA hybridization into a readily detectable electric signal by means of a conformational change of DNA stem-loop structure. Using this platform, we successfully demonstrate the detection of as low as 100 nM E. coli sequences and the automatic interpretation and mapping of the detection results via a mobile application. PMID:27170858

Dual-colored graphene quantum dots-labeled nanoprobes/graphene oxide: functional carbon materials for respective and simultaneous detection of DNA and thrombin

NASA Astrophysics Data System (ADS)

Qian, Zhao Sheng; Shan, Xiao Yue; Chai, Lu Jing; Chen, Jian Rong; Feng, Hui

2014-10-01

Convenient and simultaneous detection of multiple biomarkers such as DNA and proteins with biocompatible materials and good analytical performance still remains a challenge. Herein, we report the respective and simultaneous detection of DNA and bovine α-thrombin (thrombin) entirely based on biocompatible carbon materials through a specially designed fluorescence on-off-on process. Colorful fluorescence, high emission efficiency, good photostability and excellent compatibility enables graphene quantum dots (GQDs) as the best choice for fluorophores in bioprobes, and thus two-colored GQDs as labeling fluorophores were chemically bonded with specific oligonucleotide sequence and aptamer to prepare two probes targeting the DNA and thrombin, respectively. Each probe can be assembled on the graphene oxide (GO) platform spontaneously by π-π stacking and electrostatic attraction; as a result, fast electron transfer in the assembly efficiently quenches the fluorescence of probe. The presence of DNA or thrombin can trigger the self-recognition between capturing a nucleotide sequence and its target DNA or between thrombin and its aptamer due to their specific hybridization and duplex DNA structures or the formation of apatamer-substrate complex, which is taken advantage of in order to achieve a separate quantitative analysis of DNA and thrombin. A dual-functional biosensor for simultaneous detection of DNA and thrombin was also constructed by self-assembly of two probes with distinct colors and GO platform, and was further evaluated with the presence of various concentrations of DNA and thrombin. Both biosensors serving as a general detection model for multiple species exhibit outstanding analytical performance, and are expected to be applied in vivo because of the excellent biocompatibility of their used materials.

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs).

PubMed

Yu, Shann S; Scherer, Randy L; Ortega, Ryan A; Bell, Charleson S; O'Neil, Conlin P; Hubbell, Jeffrey A; Giorgio, Todd D

2011-02-27

Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques.

BioBarcode: a general DNA barcoding database and server platform for Asian biodiversity resources.

PubMed

Lim, Jeongheui; Kim, Sang-Yoon; Kim, Sungmin; Eo, Hae-Seok; Kim, Chang-Bae; Paek, Woon Kee; Kim, Won; Bhak, Jong

2009-12-03

DNA barcoding provides a rapid, accurate, and standardized method for species-level identification using short DNA sequences. Such a standardized identification method is useful for mapping all the species on Earth, particularly when DNA sequencing technology is cheaply available. There are many nations in Asia with many biodiversity resources that need to be mapped and registered in databases. We have built a general DNA barcode data processing system, BioBarcode, with open source software - which is a general purpose database and server. It uses mySQL RDBMS 5.0, BLAST2, and Apache httpd server. An exemplary database of BioBarcode has around 11,300 specimen entries (including GenBank data) and registers the biological species to map their genetic relationships. The BioBarcode database contains a chromatogram viewer which improves the performance in DNA sequence analyses. Asia has a very high degree of biodiversity and the BioBarcode database server system aims to provide an efficient bioinformatics protocol that can be freely used by Asian researchers and research organizations interested in DNA barcoding. The BioBarcode promotes the rapid acquisition of biological species DNA sequence data that meet global standards by providing specialized services, and provides useful tools that will make barcoding cheaper and faster in the biodiversity community such as standardization, depository, management, and analysis of DNA barcode data. The system can be downloaded upon request, and an exemplary server has been constructed with which to build an Asian biodiversity system http://www.asianbarcode.org.

A Single Electrochemical Probe Used for Analysis of Multiple Nucleic Acid Sequences

PubMed Central

Mills, Dawn M.; Calvo-Marzal, Percy; Pinzon, Jeffer M.; Armas, Stephanie; Kolpashchikov, Dmitry M.; Chumbimuni-Torres, Karin Y.

2017-01-01

Electrochemical hybridization sensors have been explored extensively for analysis of specific nucleic acids. However, commercialization of the platform is hindered by the need for attachment of separate oligonucleotide probes complementary to a RNA or DNA target to an electrode’s surface. Here we demonstrate that a single probe can be used to analyze several nucleic acid targets with high selectivity and low cost. The universal electrochemical four-way junction (4J)-forming (UE4J) sensor consists of a universal DNA stem-loop (USL) probe attached to the electrode’s surface and two adaptor strands (m and f) which hybridize to the USL probe and the analyte to form a 4J associate. The m adaptor strand was conjugated with a methylene blue redox marker for signal ON sensing and monitored using square wave voltammetry. We demonstrated that a single sensor can be used for detection of several different DNA/RNA sequences and can be regenerated in 30 seconds by a simple water rinse. The UE4J sensor enables a high selectivity by recognition of a single base substitution, even at room temperature. The UE4J sensor opens a venue for a re-useable universal platform that can be adopted at low cost for the analysis of DNA or RNA targets. PMID:29371782

DNA impedance biosensor for detection of cancer, TP53 gene mutation, based on gold nanoparticles/aligned carbon nanotubes modified electrode.

PubMed

Fayazfar, H; Afshar, A; Dolati, M; Dolati, A

2014-07-11

For the first time, a new platform based on electrochemical growth of Au nanoparticles on aligned multi-walled carbon nanotubes (A-MWCNT) was developed for sensitive lable-free DNA detection of the TP53 gene mutation, one of the most popular genes in cancer research. Electrochemical impedance spectroscopy (EIS) was used to monitor the sequence-specific DNA hybridization events related to TP53 gene. Compared to the bare Ta or MWCNT/Ta electrodes, the synergistic interactions of vertically aligned MWCNT array and gold nanoparticles at modified electrode could improve the density of the probe DNA attachment and resulting the sensitivity of the DNA sensor greatly. Using EIS, over the extended DNA concentration range, the change of charge transfer resistance was found to have a linear relationship in respect to the logarithm of the complementary oligonucleotides sequence concentrations in the wide range of 1.0×10(-15)-1.0×10(-7)M, with a detection limit of 1.0×10(-17)M (S/N=3). The prepared sensor also showed good stability (14 days), reproducibility (RSD=2.1%) and could be conveniently regenerated via dehybridization in hot water. The significant improvement in sensitivity illustrates that combining gold nanoparticles with the on-site fabricated aligned MWCNT array represents a promising platform for achieving sensitive biosensor for fast mutation screening related to most human cancer types. Copyright © 2014. Published by Elsevier B.V.

Sensitive DNA detection and SNP discrimination using ultrabright SERS nanorattles and magnetic beads for malaria diagnostics.

PubMed

Ngo, Hoan T; Gandra, Naveen; Fales, Andrew M; Taylor, Steve M; Vo-Dinh, Tuan

2016-07-15

One of the major obstacles to implement nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is the lack of sensitive and practical DNA detection methods that can be seamlessly integrated into portable platforms. Herein we present a sensitive yet simple DNA detection method using a surface-enhanced Raman scattering (SERS) nanoplatform: the ultrabright SERS nanorattle. The method, referred to as the nanorattle-based method, involves sandwich hybridization of magnetic beads that are loaded with capture probes, target sequences, and ultrabright SERS nanorattles that are loaded with reporter probes. Upon hybridization, a magnet was applied to concentrate the hybridization sandwiches at a detection spot for SERS measurements. The ultrabright SERS nanorattles, composed of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for signal detection. Using this method, a specific DNA sequence of the malaria parasite Plasmodium falciparum could be detected with a detection limit of approximately 100 attomoles. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. These test models demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. Furthermore, the method's simplicity makes it a suitable candidate for integration into portable platforms for POC and in resource-limited settings applications. Copyright © 2016. Published by Elsevier B.V.

The Essential Component in DNA-Based Information Storage System: Robust Error-Tolerating Module

PubMed Central

Yim, Aldrin Kay-Yuen; Yu, Allen Chi-Shing; Li, Jing-Woei; Wong, Ada In-Chun; Loo, Jacky F. C.; Chan, King Ming; Kong, S. K.; Yip, Kevin Y.; Chan, Ting-Fung

2014-01-01

The size of digital data is ever increasing and is expected to grow to 40,000 EB by 2020, yet the estimated global information storage capacity in 2011 is <300 EB, indicating that most of the data are transient. DNA, as a very stable nano-molecule, is an ideal massive storage device for long-term data archive. The two most notable illustrations are from Church et al. and Goldman et al., whose approaches are well-optimized for most sequencing platforms – short synthesized DNA fragments without homopolymer. Here, we suggested improvements on error handling methodology that could enable the integration of DNA-based computational process, e.g., algorithms based on self-assembly of DNA. As a proof of concept, a picture of size 438 bytes was encoded to DNA with low-density parity-check error-correction code. We salvaged a significant portion of sequencing reads with mutations generated during DNA synthesis and sequencing and successfully reconstructed the entire picture. A modular-based programing framework – DNAcodec with an eXtensible Markup Language-based data format was also introduced. Our experiments demonstrated the practicability of long DNA message recovery with high error tolerance, which opens the field to biocomputing and synthetic biology. PMID:25414846

Analysis of Pre-Analytic Factors Affecting the Success of Clinical Next-Generation Sequencing of Solid Organ Malignancies.

PubMed

Chen, Hui; Luthra, Rajyalakshmi; Goswami, Rashmi S; Singh, Rajesh R; Roy-Chowdhuri, Sinchita

2015-08-28

Application of next-generation sequencing (NGS) technology to routine clinical practice has enabled characterization of personalized cancer genomes to identify patients likely to have a response to targeted therapy. The proper selection of tumor sample for downstream NGS based mutational analysis is critical to generate accurate results and to guide therapeutic intervention. However, multiple pre-analytic factors come into play in determining the success of NGS testing. In this review, we discuss pre-analytic requirements for AmpliSeq PCR-based sequencing using Ion Torrent Personal Genome Machine (PGM) (Life Technologies), a NGS sequencing platform that is often used by clinical laboratories for sequencing solid tumors because of its low input DNA requirement from formalin fixed and paraffin embedded tissue. The success of NGS mutational analysis is affected not only by the input DNA quantity but also by several other factors, including the specimen type, the DNA quality, and the tumor cellularity. Here, we review tissue requirements for solid tumor NGS based mutational analysis, including procedure types, tissue types, tumor volume and fraction, decalcification, and treatment effects.

Microsatellite DNA capture from enriched libraries.

PubMed

Gonzalez, Elena G; Zardoya, Rafael

2013-01-01

Microsatellites are DNA sequences of tandem repeats of one to six nucleotides, which are highly polymorphic, and thus the molecular markers of choice in many kinship, population genetic, and conservation studies. There have been significant technical improvements since the early methods for microsatellite isolation were developed, and today the most common procedures take advantage of the hybrid capture methods of enriched-targeted microsatellite DNA. Furthermore, recent advents in sequencing technologies (i.e., next-generation sequencing, NGS) have fostered the mining of microsatellite markers in non-model organisms, affording a cost-effective way of obtaining a large amount of sequence data potentially useful for loci characterization. The rapid improvements of NGS platforms together with the increase in available microsatellite information open new avenues to the understanding of the evolutionary forces that shape genetic structuring in wild populations. Here, we provide detailed methodological procedures for microsatellite isolation based on the screening of GT microsatellite-enriched libraries, either by cloning and Sanger sequencing of positive clones or by direct NGS. Guides for designing new species-specific primers and basic genotyping are also given.

The challenges of sequencing by synthesis.

PubMed

Fuller, Carl W; Middendorf, Lyle R; Benner, Steven A; Church, George M; Harris, Timothy; Huang, Xiaohua; Jovanovich, Stevan B; Nelson, John R; Schloss, Jeffery A; Schwartz, David C; Vezenov, Dmitri V

2009-11-01

DNA sequencing-by-synthesis (SBS) technology, using a polymerase or ligase enzyme as its core biochemistry, has already been incorporated in several second-generation DNA sequencing systems with significant performance. Notwithstanding the substantial success of these SBS platforms, challenges continue to limit the ability to reduce the cost of sequencing a human genome to $100,000 or less. Achieving dramatically reduced cost with enhanced throughput and quality will require the seamless integration of scientific and technological effort across disciplines within biochemistry, chemistry, physics and engineering. The challenges include sample preparation, surface chemistry, fluorescent labels, optimizing the enzyme-substrate system, optics, instrumentation, understanding tradeoffs of throughput versus accuracy, and read-length/phasing limitations. By framing these challenges in a manner accessible to a broad community of scientists and engineers, we hope to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.

BioWires: Conductive DNA Nanowires in a Computationally-Optimized, Synthetic Biological Platform for Nanoelectronic Fabrication

NASA Technical Reports Server (NTRS)

Vecchioni, Simon; Toomey, Emily; Capece, Mark C.; Rothschild, Lynn; Wind, Shalom

2017-01-01

DNA is an ideal template for a biological nanowire-it has a linear structure several atoms thick; it possesses addressable nucleobase geometry that can be precisely defined; and it is massively scalable into branched networks. Until now, the drawback of DNA as a conducting nanowire been, simply put, its low conductance. To address this deficiency, we extensively characterize a chemical variant of canonical DNA that exploits the affinity of natural cytosine bases for silver ions. We successfully construct chains of single silver ions inside double-stranded DNA, confirm the basic dC-Ag+-dC bond geometry and kinetics, and show length-tunability dependent on mismatch distribution, ion availability and enzyme activity. An analysis of the absorbance spectra of natural DNA and silver-binding, poly-cytosine DNA demonstrates the heightened thermostability of the ion chain and its resistance to aqueous stresses such as precipitation, dialysis and forced reduction. These chemically critical traits lend themselves to an increase in electrical conductivity of over an order of magnitude for 11-base silver-paired duplexes over natural strands when assayed by STM break junction. We further construct and implement a genetic pathway in the E. coli bacterium for the biosynthesis of highly ionizable DNA sequences. Toward future circuits, we construct a model of transcription network architectures to determine the most efficient and robust connectivity for cell-based fabrication, and we perform sequence optimization with a genetic algorithm to identify oligonucleotides robust to changes in the base-pairing energy landscape. We propose that this system will serve as a synthetic biological fabrication platform for more complex DNA nanotechnology and nanoelectronics with applications to deep space and low resource environments.

Dissecting enzyme function with microfluidic-based deep mutational scanning.

PubMed

Romero, Philip A; Tran, Tuan M; Abate, Adam R

2015-06-09

Natural enzymes are incredibly proficient catalysts, but engineering them to have new or improved functions is challenging due to the complexity of how an enzyme's sequence relates to its biochemical properties. Here, we present an ultrahigh-throughput method for mapping enzyme sequence-function relationships that combines droplet microfluidic screening with next-generation DNA sequencing. We apply our method to map the activity of millions of glycosidase sequence variants. Microfluidic-based deep mutational scanning provides a comprehensive and unbiased view of the enzyme function landscape. The mapping displays expected patterns of mutational tolerance and a strong correspondence to sequence variation within the enzyme family, but also reveals previously unreported sites that are crucial for glycosidase function. We modified the screening protocol to include a high-temperature incubation step, and the resulting thermotolerance landscape allowed the discovery of mutations that enhance enzyme thermostability. Droplet microfluidics provides a general platform for enzyme screening that, when combined with DNA-sequencing technologies, enables high-throughput mapping of enzyme sequence space.

Assessing macroinvertebrate biodiversity in freshwater ecosystems: Advances and challenges in dna-based approaches

USGS Publications Warehouse

Pfrender, M.E.; Ferrington, L.C.; Hawkins, C.P.; Hartzell, P.L.; Bagley, M.; Jackson, S.; Courtney, G.W.; Larsen, D.P.; Creutzburg, B.R.; Levesque, C.A.; Epler, J.H.; Morse, J.C.; Fend, S.; Petersen, M.J.; Ruiter, D.; Schindel, D.; Whiting, M.

2010-01-01

Assessing the biodiversity of macroinvertebrate fauna in freshwater ecosystems is an essential component of both basic ecological inquiry and applied ecological assessments. Aspects of taxonomic diversity and composition in freshwater communities are widely used to quantify water quality and measure the efficacy of remediation and restoration efforts. The accuracy and precision of biodiversity assessments based on standard morphological identifications are often limited by taxonomic resolution and sample size. Morphologically based identifications are laborious and costly, significantly constraining the sample sizes that can be processed. We suggest that the development of an assay platform based on DNA signatures will increase the precision and ease of quantifying biodiversity in freshwater ecosystems. Advances in this area will be particularly relevant for benthic and planktonic invertebrates, which are often monitored by regulatory agencies. Adopting a genetic assessment platform will alleviate some of the current limitations to biodiversity assessment strategies. We discuss the benefits and challenges associated with DNA-based assessments and the methods that are currently available. As recent advances in microarray and next-generation sequencing technologies will facilitate a transition to DNA-based assessment approaches, future research efforts should focus on methods for data collection, assay platform development, establishing linkages between DNA signatures and well-resolved taxonomies, and bioinformatics. ?? 2010 by The University of Chicago Press.

Benchmarking of TALE- and CRISPR/dCas9-Based Transcriptional Regulators in Mammalian Cells for the Construction of Synthetic Genetic Circuits.

PubMed

Lebar, Tina; Jerala, Roman

2016-10-21

Transcriptional activator-like effector (TALE)- and CRISPR/Cas9-based designable recognition domains represent a technological breakthrough not only for genome editing but also for building designed genetic circuits. Both platforms are able to target rarely occurring DNA segments, even within complex genomes. TALE and dCas9 domains, genetically fused to transcriptional regulatory domains, can be used for the construction of engineered logic circuits. Here we benchmarked the performance of the two platforms, targeting the same DNA sequences, to compare their advantages for the construction of designed circuits in mammalian cells. Optimal targeting strands for repression and activation of dCas9-based designed transcription factors were identified; both platforms exhibited good orthogonality and were used to construct functionally complete NOR gates. Although the CRISPR/dCas9 system is clearly easier to construct, TALE-based activators were significantly stronger, and the TALE-based platform performed better, especially for the construction of layered circuits.

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization.

PubMed

Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

2017-01-01

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.

MEGGASENSE - The Metagenome/Genome Annotated Sequence Natural Language Search Engine: A Platform for 
the Construction of Sequence Data Warehouses.

PubMed

Gacesa, Ranko; Zucko, Jurica; Petursdottir, Solveig K; Gudmundsdottir, Elisabet Eik; Fridjonsson, Olafur H; Diminic, Janko; Long, Paul F; Cullum, John; Hranueli, Daslav; Hreggvidsson, Gudmundur O; Starcevic, Antonio

2017-06-01

The MEGGASENSE platform constructs relational databases of DNA or protein sequences. The default functional analysis uses 14 106 hidden Markov model (HMM) profiles based on sequences in the KEGG database. The Solr search engine allows sophisticated queries and a BLAST search function is also incorporated. These standard capabilities were used to generate the SCATT database from the predicted proteome of Streptomyces cattleya . The implementation of a specialised metagenome database (AMYLOMICS) for bioprospecting of carbohydrate-modifying enzymes is described. In addition to standard assembly of reads, a novel 'functional' assembly was developed, in which screening of reads with the HMM profiles occurs before the assembly. The AMYLOMICS database incorporates additional HMM profiles for carbohydrate-modifying enzymes and it is illustrated how the combination of HMM and BLAST analyses helps identify interesting genes. A variety of different proteome and metagenome databases have been generated by MEGGASENSE.

Gallium plasmonic nanoparticles for label-free DNA and single nucleotide polymorphism sensing

NASA Astrophysics Data System (ADS)

Marín, Antonio García; García-Mendiola, Tania; Bernabeu, Cristina Navio; Hernández, María Jesús; Piqueras, Juan; Pau, Jose Luis; Pariente, Félix; Lorenzo, Encarnación

2016-05-01

A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori onto GaNP/Si substrates; complementary target sequences of Helicobacter pylori can be quantified over the range of 10 pM to 3.0 nM with a detection limit of 6.0 pM and a linear correlation coefficient of R2 = 0.990. The selectivity of the device allows the detection of a single nucleotide polymorphism (SNP) in a specific sequence of Helicobacter pylori, without the need for a hybridization suppressor in solution such as formamide. Furthermore, it also allows the detection of this sequence in the presence of other pathogens, such as Escherichia coli in the sample. The broad applicability of the system was demonstrated by the detection of a specific gene mutation directly associated with cystic fibrosis in large genomic DNA isolated from blood cells.A label-free DNA and single nucleotide polymorphism (SNP) sensing method is described. It is based on the use of the pseudodielectric function of gallium plasmonic nanoparticles (GaNPs) deposited on Si (100) substrates under reversal of the polarization handedness condition. Under this condition, the pseudodielectric function is extremely sensitive to changes in the surrounding medium of the nanoparticle surface providing an excellent sensing platform competitive to conventional surface plasmon resonance. DNA sensing has been carried out by immobilizing a thiolated capture probe sequence from Helicobacter pylori onto GaNP/Si substrates; complementary target sequences of Helicobacter pylori can be quantified over the range of 10 pM to 3.0 nM with a detection limit of 6.0 pM and a linear correlation coefficient of R2 = 0.990. The selectivity of the device allows the detection of a single nucleotide polymorphism (SNP) in a specific sequence of Helicobacter pylori, without the need for a hybridization suppressor in solution such as formamide. Furthermore, it also allows the detection of this sequence in the presence of other pathogens, such as Escherichia coli in the sample. The broad applicability of the system was demonstrated by the detection of a specific gene mutation directly associated with cystic fibrosis in large genomic DNA isolated from blood cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr00926c

Combined Targeted DNA Sequencing in Non-Small Cell Lung Cancer (NSCLC) Using UNCseq and NGScopy, and RNA Sequencing Using UNCqeR for the Detection of Genetic Aberrations in NSCLC

PubMed Central

Walter, Vonn; Patel, Nirali M.; Eberhard, David A.; Hayward, Michele C.; Salazar, Ashley H.; Jo, Heejoon; Soloway, Matthew G.; Wilkerson, Matthew D.; Parker, Joel S.; Yin, Xiaoying; Zhang, Guosheng; Siegel, Marni B.; Rosson, Gary B.; Earp, H. Shelton; Sharpless, Norman E.; Gulley, Margaret L.; Weck, Karen E.

2015-01-01

The recent FDA approval of the MiSeqDx platform provides a unique opportunity to develop targeted next generation sequencing (NGS) panels for human disease, including cancer. We have developed a scalable, targeted panel-based assay termed UNCseq, which involves a NGS panel of over 200 cancer-associated genes and a standardized downstream bioinformatics pipeline for detection of single nucleotide variations (SNV) as well as small insertions and deletions (indel). In addition, we developed a novel algorithm, NGScopy, designed for samples with sparse sequencing coverage to detect large-scale copy number variations (CNV), similar to human SNP Array 6.0 as well as small-scale intragenic CNV. Overall, we applied this assay to 100 snap-frozen lung cancer specimens lacking same-patient germline DNA (07–0120 tissue cohort) and validated our results against Sanger sequencing, SNP Array, and our recently published integrated DNA-seq/RNA-seq assay, UNCqeR, where RNA-seq of same-patient tumor specimens confirmed SNV detected by DNA-seq, if RNA-seq coverage depth was adequate. In addition, we applied the UNCseq assay on an independent lung cancer tumor tissue collection with available same-patient germline DNA (11–1115 tissue cohort) and confirmed mutations using assays performed in a CLIA-certified laboratory. We conclude that UNCseq can identify SNV, indel, and CNV in tumor specimens lacking germline DNA in a cost-efficient fashion. PMID:26076459

Fragmentation of contaminant and endogenous DNA in ancient samples determined by shotgun sequencing; prospects for human palaeogenomics.

PubMed

García-Garcerà, Marc; Gigli, Elena; Sanchez-Quinto, Federico; Ramirez, Oscar; Calafell, Francesc; Civit, Sergi; Lalueza-Fox, Carles

2011-01-01

Despite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks. METHODOLOGY/PRINCIPALS FINDINGS: To investigate whether this pattern is present in ancient remains from a temperate environment, we have 454-FLX pyrosequenced different samples dated between 5,500 and 49,000 years ago: a bone from an extinct goat (Myotragus balearicus) that was treated with a depurinating agent (bleach), an Iberian lynx bone not subjected to any treatment, a human Neolithic sample from Barcelona (Spain), and a Neandertal sample from the El Sidrón site (Asturias, Spain). The efficiency of retrieval of endogenous sequences is below 1% in all cases. We have used the non-human samples to identify human sequences (0.35 and 1.4%, respectively), that we positively know are contaminants. We observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences. Furthermore, the nucleotide composition pattern observed in 5' and 3' ends of contaminant sequences is much more complex than the flat pattern previously described in some Neandertal contaminants. Although much research on samples with known contaminant histories is needed, our results suggest that endogenous and contaminant sequences cannot be distinguished by the fragmentation pattern alone.

An electrochemical sensing platform based on local repression of electrolyte diffusion for single-step, reagentless, sensitive detection of a sequence-specific DNA-binding protein.

PubMed

Zhang, Yun; Liu, Fang; Nie, Jinfang; Jiang, Fuyang; Zhou, Caibin; Yang, Jiani; Fan, Jinlong; Li, Jianping

2014-05-07

In this paper, we report for the first time an electrochemical biosensor for single-step, reagentless, and picomolar detection of a sequence-specific DNA-binding protein using a double-stranded, electrode-bound DNA probe terminally modified with a redox active label close to the electrode surface. This new methodology is based upon local repression of electrolyte diffusion associated with protein-DNA binding that leads to reduction of the electrochemical response of the label. In the proof-of-concept study, the resulting electrochemical biosensor was quantitatively sensitive to the concentrations of the TATA binding protein (TBP, a model analyte) ranging from 40 pM to 25.4 nM with an estimated detection limit of ∼10.6 pM (∼80 to 400-fold improvement on the detection limit over previous electrochemical analytical systems).

Next Generation MUT-MAP, a High-Sensitivity High-Throughput Microfluidics Chip-Based Mutation Analysis Panel

PubMed Central

Patel, Rajesh; Tsan, Alison; Sumiyoshi, Teiko; Fu, Ling; Desai, Rupal; Schoenbrunner, Nancy; Myers, Thomas W.; Bauer, Keith; Smith, Edward; Raja, Rajiv

2014-01-01

Molecular profiling of tumor tissue to detect alterations, such as oncogenic mutations, plays a vital role in determining treatment options in oncology. Hence, there is an increasing need for a robust and high-throughput technology to detect oncogenic hotspot mutations. Although commercial assays are available to detect genetic alterations in single genes, only a limited amount of tissue is often available from patients, requiring multiplexing to allow for simultaneous detection of mutations in many genes using low DNA input. Even though next-generation sequencing (NGS) platforms provide powerful tools for this purpose, they face challenges such as high cost, large DNA input requirement, complex data analysis, and long turnaround times, limiting their use in clinical settings. We report the development of the next generation mutation multi-analyte panel (MUT-MAP), a high-throughput microfluidic, panel for detecting 120 somatic mutations across eleven genes of therapeutic interest (AKT1, BRAF, EGFR, FGFR3, FLT3, HRAS, KIT, KRAS, MET, NRAS, and PIK3CA) using allele-specific PCR (AS-PCR) and Taqman technology. This mutation panel requires as little as 2 ng of high quality DNA from fresh frozen or 100 ng of DNA from formalin-fixed paraffin-embedded (FFPE) tissues. Mutation calls, including an automated data analysis process, have been implemented to run 88 samples per day. Validation of this platform using plasmids showed robust signal and low cross-reactivity in all of the newly added assays and mutation calls in cell line samples were found to be consistent with the Catalogue of Somatic Mutations in Cancer (COSMIC) database allowing for direct comparison of our platform to Sanger sequencing. High correlation with NGS when compared to the SuraSeq500 panel run on the Ion Torrent platform in a FFPE dilution experiment showed assay sensitivity down to 0.45%. This multiplexed mutation panel is a valuable tool for high-throughput biomarker discovery in personalized medicine and cancer drug development. PMID:24658394

Measurement of fetal fraction in cell-free DNA from maternal plasma using a panel of insertion/deletion polymorphisms.

PubMed

Barrett, Angela N; Xiong, Li; Tan, Tuan Z; Advani, Henna V; Hua, Rui; Laureano-Asibal, Cecille; Soong, Richie; Biswas, Arijit; Nagarajan, Niranjan; Choolani, Mahesh

2017-01-01

Cell-free DNA from maternal plasma can be used for non-invasive prenatal testing for aneuploidies and single gene disorders, and also has applications as a biomarker for monitoring high-risk pregnancies, such as those at risk of pre-eclampsia. On average, the fractional cell-free fetal DNA concentration in plasma is approximately 15%, but can vary from less than 4% to greater than 30%. Although quantification of cell-free fetal DNA is straightforward in the case of a male fetus, there is no universal fetal marker; in a female fetus measurement is more challenging. We have developed a panel of multiplexed insertion/deletion polymorphisms that can measure fetal fraction in all pregnancies in a simple, targeted sequencing reaction. A multiplex panel of primers was designed for 35 indels plus a ZFX/ZFY amplicon. cfDNA was extracted from plasma from 157 pregnant women, and maternal genomic DNA was extracted for 20 of these samples for panel validation. Sixty-one samples from pregnancies with a male fetus were subjected to whole genome sequencing on the Ion Proton sequencing platform, and fetal fraction derived from Y chromosome counts was compared to fetal fraction measured using the indel panel. A total of 157 cell-free DNA samples were sequenced using the indel panel, and informativity was assessed, along with the proportion of fetal DNA. Using gDNA we optimised the indel panel, removing amplicons giving rise to PCR bias. Good correlation was found between fetal fraction using indels and using whole genome sequencing of the Y chromosome (Spearmans r = 0.69). A median of 12 indels were informative per sample. The indel panel was informative in 157/157 cases (mean fetal fraction 14.4% (±0.58%)). Using our targeted next generation sequencing panel we can readily assess the fetal DNA percentage in male and female pregnancies.

Measurement of fetal fraction in cell-free DNA from maternal plasma using a panel of insertion/deletion polymorphisms

PubMed Central

Xiong, Li; Tan, Tuan Z.; Advani, Henna V.; Hua, Rui; Laureano-Asibal, Cecille; Soong, Richie; Biswas, Arijit; Nagarajan, Niranjan; Choolani, Mahesh

2017-01-01

Objective Cell-free DNA from maternal plasma can be used for non-invasive prenatal testing for aneuploidies and single gene disorders, and also has applications as a biomarker for monitoring high-risk pregnancies, such as those at risk of pre-eclampsia. On average, the fractional cell-free fetal DNA concentration in plasma is approximately 15%, but can vary from less than 4% to greater than 30%. Although quantification of cell-free fetal DNA is straightforward in the case of a male fetus, there is no universal fetal marker; in a female fetus measurement is more challenging. We have developed a panel of multiplexed insertion/deletion polymorphisms that can measure fetal fraction in all pregnancies in a simple, targeted sequencing reaction. Methods A multiplex panel of primers was designed for 35 indels plus a ZFX/ZFY amplicon. cfDNA was extracted from plasma from 157 pregnant women, and maternal genomic DNA was extracted for 20 of these samples for panel validation. Sixty-one samples from pregnancies with a male fetus were subjected to whole genome sequencing on the Ion Proton sequencing platform, and fetal fraction derived from Y chromosome counts was compared to fetal fraction measured using the indel panel. A total of 157 cell-free DNA samples were sequenced using the indel panel, and informativity was assessed, along with the proportion of fetal DNA. Results Using gDNA we optimised the indel panel, removing amplicons giving rise to PCR bias. Good correlation was found between fetal fraction using indels and using whole genome sequencing of the Y chromosome (Spearmans r = 0.69). A median of 12 indels were informative per sample. The indel panel was informative in 157/157 cases (mean fetal fraction 14.4% (±0.58%)). Conclusions Using our targeted next generation sequencing panel we can readily assess the fetal DNA percentage in male and female pregnancies. PMID:29084245

DNA nanotechnology: new adventures for an old warhorse.

PubMed

Zakeri, Bijan; Lu, Timothy K

2015-10-01

As the blueprint of life, the natural exploits of DNA are admirable. However, DNA should not only be viewed within a biological context. It is an elegantly simple yet functionally complex chemical polymer with properties that make it an ideal platform for engineering new nanotechnologies. Rapidly advancing synthesis and sequencing technologies are enabling novel unnatural applications for DNA beyond the realm of genetics. Here we explore the chemical biology of DNA nanotechnology for emerging applications in communication and digital data storage. Early studies of DNA as an alternative to magnetic and optical storage mediums have not only been promising, but have demonstrated the potential of DNA to revolutionize the way we interact with digital data in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

DNA nanotechnology: new adventures for an old warhorse

PubMed Central

Zakeri, Bijan; Lu, Timothy K.

2016-01-01

As the blueprint of life, the natural exploits of DNA are admirable. However, DNA should not only be viewed within a biological context. It is an elegantly simple yet functionally complex chemical polymer with properties that make it an ideal platform for engineering new nanotechnologies. Rapidly advancing synthesis and sequencing technologies are enabling novel unnatural applications for DNA beyond the realm of genetics. Here we explore the chemical biology of DNA nanotechnology for emerging applications in communication and digital data storage. Early studies of DNA as an alternative to magnetic and optical storage mediums have not only been promising, but have demonstrated the potential of DNA to revolutionize the way we interact with digital data in the future. PMID:26056949

A novel ultra high-throughput 16S rRNA gene amplicon sequencing library preparation method for the Illumina HiSeq platform.

PubMed

de Muinck, Eric J; Trosvik, Pål; Gilfillan, Gregor D; Hov, Johannes R; Sundaram, Arvind Y M

2017-07-06

Advances in sequencing technologies and bioinformatics have made the analysis of microbial communities almost routine. Nonetheless, the need remains to improve on the techniques used for gathering such data, including increasing throughput while lowering cost and benchmarking the techniques so that potential sources of bias can be better characterized. We present a triple-index amplicon sequencing strategy to sequence large numbers of samples at significantly lower c ost and in a shorter timeframe compared to existing methods. The design employs a two-stage PCR protocol, incorpo rating three barcodes to each sample, with the possibility to add a fourth-index. It also includes heterogeneity spacers to overcome low complexity issues faced when sequencing amplicons on Illumina platforms. The library preparation method was extensively benchmarked through analysis of a mock community in order to assess biases introduced by sample indexing, number of PCR cycles, and template concentration. We further evaluated the method through re-sequencing of a standardized environmental sample. Finally, we evaluated our protocol on a set of fecal samples from a small cohort of healthy adults, demonstrating good performance in a realistic experimental setting. Between-sample variation was mainly related to batch effects, such as DNA extraction, while sample indexing was also a significant source of bias. PCR cycle number strongly influenced chimera formation and affected relative abundance estimates of species with high GC content. Libraries were sequenced using the Illumina HiSeq and MiSeq platforms to demonstrate that this protocol is highly scalable to sequence thousands of samples at a very low cost. Here, we provide the most comprehensive study of performance and bias inherent to a 16S rRNA gene amplicon sequencing method to date. Triple-indexing greatly reduces the number of long custom DNA oligos required for library preparation, while the inclusion of variable length heterogeneity spacers minimizes the need for PhiX spike-in. This design results in a significant cost reduction of highly multiplexed amplicon sequencing. The biases we characterize highlight the need for highly standardized protocols. Reassuringly, we find that the biological signal is a far stronger structuring factor than the various sources of bias.

A Review on the Applications of Next Generation Sequencing Technologies as Applied to Food-Related Microbiome Studies

PubMed Central

Cao, Yu; Fanning, Séamus; Proos, Sinéad; Jordan, Kieran; Srikumar, Shabarinath

2017-01-01

The development of next generation sequencing (NGS) techniques has enabled researchers to study and understand the world of microorganisms from broader and deeper perspectives. The contemporary advances in DNA sequencing technologies have not only enabled finer characterization of bacterial genomes but also provided deeper taxonomic identification of complex microbiomes which in its genomic essence is the combined genetic material of the microorganisms inhabiting an environment, whether the environment be a particular body econiche (e.g., human intestinal contents) or a food manufacturing facility econiche (e.g., floor drain). To date, 16S rDNA sequencing, metagenomics and metatranscriptomics are the three basic sequencing strategies used in the taxonomic identification and characterization of food-related microbiomes. These sequencing strategies have used different NGS platforms for DNA and RNA sequence identification. Traditionally, 16S rDNA sequencing has played a key role in understanding the taxonomic composition of a food-related microbiome. Recently, metagenomic approaches have resulted in improved understanding of a microbiome by providing a species-level/strain-level characterization. Further, metatranscriptomic approaches have contributed to the functional characterization of the complex interactions between different microbial communities within a single microbiome. Many studies have highlighted the use of NGS techniques in investigating the microbiome of fermented foods. However, the utilization of NGS techniques in studying the microbiome of non-fermented foods are limited. This review provides a brief overview of the advances in DNA sequencing chemistries as the technology progressed from first, next and third generations and highlights how NGS provided a deeper understanding of food-related microbiomes with special focus on non-fermented foods. PMID:29033905

Epigenetics of prostate cancer.

PubMed

McKee, Tawnya C; Tricoli, James V

2015-01-01

The introduction of novel technologies that can be applied to the investigation of the molecular underpinnings of human cancer has allowed for new insights into the mechanisms associated with tumor development and progression. They have also advanced the diagnosis, prognosis and treatment of cancer. These technologies include microarray and other analysis methods for the generation of large-scale gene expression data on both mRNA and miRNA, next-generation DNA sequencing technologies utilizing a number of platforms to perform whole genome, whole exome, or targeted DNA sequencing to determine somatic mutational differences and gene rearrangements, and a variety of proteomic analysis platforms including liquid chromatography/mass spectrometry (LC/MS) analysis to survey alterations in protein profiles in tumors. One other important advancement has been our current ability to survey the methylome of human tumors in a comprehensive fashion through the use of sequence-based and array-based methylation analysis (Bock et al., Nat Biotechnol 28:1106-1114, 2010; Harris et al., Nat Biotechnol 28:1097-1105, 2010). The focus of this chapter is to present and discuss the evidence for key genes involved in prostate tumor development, progression, or resistance to therapy that are regulated by methylation-induced silencing.

DNApod: DNA polymorphism annotation database from next-generation sequence read archives.

PubMed

Mochizuki, Takako; Tanizawa, Yasuhiro; Fujisawa, Takatomo; Ohta, Tazro; Nikoh, Naruo; Shimizu, Tokurou; Toyoda, Atsushi; Fujiyama, Asao; Kurata, Nori; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information.

BioBarcode: a general DNA barcoding database and server platform for Asian biodiversity resources

PubMed Central

2009-01-01

Background DNA barcoding provides a rapid, accurate, and standardized method for species-level identification using short DNA sequences. Such a standardized identification method is useful for mapping all the species on Earth, particularly when DNA sequencing technology is cheaply available. There are many nations in Asia with many biodiversity resources that need to be mapped and registered in databases. Results We have built a general DNA barcode data processing system, BioBarcode, with open source software - which is a general purpose database and server. It uses mySQL RDBMS 5.0, BLAST2, and Apache httpd server. An exemplary database of BioBarcode has around 11,300 specimen entries (including GenBank data) and registers the biological species to map their genetic relationships. The BioBarcode database contains a chromatogram viewer which improves the performance in DNA sequence analyses. Conclusion Asia has a very high degree of biodiversity and the BioBarcode database server system aims to provide an efficient bioinformatics protocol that can be freely used by Asian researchers and research organizations interested in DNA barcoding. The BioBarcode promotes the rapid acquisition of biological species DNA sequence data that meet global standards by providing specialized services, and provides useful tools that will make barcoding cheaper and faster in the biodiversity community such as standardization, depository, management, and analysis of DNA barcode data. The system can be downloaded upon request, and an exemplary server has been constructed with which to build an Asian biodiversity system http://www.asianbarcode.org. PMID:19958506

DNApod: DNA polymorphism annotation database from next-generation sequence read archives

PubMed Central

Mochizuki, Takako; Tanizawa, Yasuhiro; Fujisawa, Takatomo; Ohta, Tazro; Nikoh, Naruo; Shimizu, Tokurou; Toyoda, Atsushi; Fujiyama, Asao; Kurata, Nori; Nagasaki, Hideki; Kaminuma, Eli; Nakamura, Yasukazu

2017-01-01

With the rapid advances in next-generation sequencing (NGS), datasets for DNA polymorphisms among various species and strains have been produced, stored, and distributed. However, reliability varies among these datasets because the experimental and analytical conditions used differ among assays. Furthermore, such datasets have been frequently distributed from the websites of individual sequencing projects. It is desirable to integrate DNA polymorphism data into one database featuring uniform quality control that is distributed from a single platform at a single place. DNA polymorphism annotation database (DNApod; http://tga.nig.ac.jp/dnapod/) is an integrated database that stores genome-wide DNA polymorphism datasets acquired under uniform analytical conditions, and this includes uniformity in the quality of the raw data, the reference genome version, and evaluation algorithms. DNApod genotypic data are re-analyzed whole-genome shotgun datasets extracted from sequence read archives, and DNApod distributes genome-wide DNA polymorphism datasets and known-gene annotations for each DNA polymorphism. This new database was developed for storing genome-wide DNA polymorphism datasets of plants, with crops being the first priority. Here, we describe our analyzed data for 679, 404, and 66 strains of rice, maize, and sorghum, respectively. The analytical methods are available as a DNApod workflow in an NGS annotation system of the DNA Data Bank of Japan and a virtual machine image. Furthermore, DNApod provides tables of links of identifiers between DNApod genotypic data and public phenotypic data. To advance the sharing of organism knowledge, DNApod offers basic and ubiquitous functions for multiple alignment and phylogenetic tree construction by using orthologous gene information. PMID:28234924

Real-Time PCR Assay To Detect Smallpox Virus

PubMed Central

Sofi Ibrahim, M.; Kulesh, David A.; Saleh, Sharron S.; Damon, Inger K.; Esposito, Joseph J.; Schmaljohn, Alan L.; Jahrling, Peter B.

2003-01-01

We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler and the LightCycler platforms. The assay may be useful for the early detection of smallpox virus infections should such infections occur as a result of a deliberate or an accidental recurrence. PMID:12904397

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision.

PubMed

Perreault, Andrea A; Venters, Bryan J

2016-12-23

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

GenomicTools: a computational platform for developing high-throughput analytics in genomics.

PubMed

Tsirigos, Aristotelis; Haiminen, Niina; Bilal, Erhan; Utro, Filippo

2012-01-15

Recent advances in sequencing technology have resulted in the dramatic increase of sequencing data, which, in turn, requires efficient management of computational resources, such as computing time, memory requirements as well as prototyping of computational pipelines. We present GenomicTools, a flexible computational platform, comprising both a command-line set of tools and a C++ API, for the analysis and manipulation of high-throughput sequencing data such as DNA-seq, RNA-seq, ChIP-seq and MethylC-seq. GenomicTools implements a variety of mathematical operations between sets of genomic regions thereby enabling the prototyping of computational pipelines that can address a wide spectrum of tasks ranging from pre-processing and quality control to meta-analyses. Additionally, the GenomicTools platform is designed to analyze large datasets of any size by minimizing memory requirements. In practical applications, where comparable, GenomicTools outperforms existing tools in terms of both time and memory usage. The GenomicTools platform (version 2.0.0) was implemented in C++. The source code, documentation, user manual, example datasets and scripts are available online at http://code.google.com/p/ibm-cbc-genomic-tools.

Theory and modeling of particles with DNA-mediated interactions

NASA Astrophysics Data System (ADS)

Licata, Nicholas A.

In recent years significant attention has been attracted to proposals which utilize DNA for nanotechnological applications. Potential applications of these ideas range from the programmable self-assembly of colloidal crystals, to biosensors and nanoparticle based drug delivery platforms. In Chapter I we introduce the system, which generically consists of colloidal particles functionalized with specially designed DNA markers. The sequence of bases on the DNA markers determines the particle type. Due to the hybridization between complementary single-stranded DNA, specific, type-dependent interactions can be introduced between particles by choosing the appropriate DNA marker sequences. In Chapter II we develop a statistical mechanical description of the aggregation and melting behavior of particles with DNA-mediated interactions. A quantitative comparison between the theory and experiments is made by calculating the experimentally observed melting profile. In Chapter III a model is proposed to describe the dynamical departure and diffusion of particles which form reversible key-lock connections. The model predicts a crossover from localized to diffusive behavior. The random walk statistics for the particles' in plane diffusion is discussed. The lateral motion is analogous to dispersive transport in disordered semiconductors, ranging from standard diffusion with a renormalized diffusion coefficient to anomalous, subdiffusive behavior. In Chapter IV we propose a method to self-assemble nanoparticle clusters using DNA scaffolds. An optimal concentration ratio is determined for the experimental implementation of our self-assembly proposal. A natural extension is discussed in Chapter V, the programmable self-assembly of nanoparticle clusters where the desired cluster geometry is encoded using DNA-mediated interactions. We determine the probability that the system self-assembles the desired cluster geometry, and discuss the connections to jamming in granular and colloidal systems. In Chapter VI we consider a nanoparticle based drug delivery platform for targeted, cell specific chemotherapy. A key-lock model is proposed to describe the results of in-vitro experiments, and the situation in-vivo is discussed. The cooperative binding, and hence the specificity to cancerous cells, is kinetically limited. The implications for optimizing the design of nanoparticle based drug delivery platforms is discussed. In Chapter VII we present prospects for future research: the connection between DNA-mediated colloidal crystallization and jamming, and the inverse problem in self-assembly.

Nanopore DNA Sequencing and Genome Assembly on the International Space Station.

PubMed

Castro-Wallace, Sarah L; Chiu, Charles Y; John, Kristen K; Stahl, Sarah E; Rubins, Kathleen H; McIntyre, Alexa B R; Dworkin, Jason P; Lupisella, Mark L; Smith, David J; Botkin, Douglas J; Stephenson, Timothy A; Juul, Sissel; Turner, Daniel J; Izquierdo, Fernando; Federman, Scot; Stryke, Doug; Somasekar, Sneha; Alexander, Noah; Yu, Guixia; Mason, Christopher E; Burton, Aaron S

2017-12-21

We evaluated the performance of the MinION DNA sequencer in-flight on the International Space Station (ISS), and benchmarked its performance off-Earth against the MinION, Illumina MiSeq, and PacBio RS II sequencing platforms in terrestrial laboratories. Samples contained equimolar mixtures of genomic DNA from lambda bacteriophage, Escherichia coli (strain K12, MG1655) and Mus musculus (female BALB/c mouse). Nine sequencing runs were performed aboard the ISS over a 6-month period, yielding a total of 276,882 reads with no apparent decrease in performance over time. From sequence data collected aboard the ISS, we constructed directed assemblies of the ~4.6 Mb E. coli genome, ~48.5 kb lambda genome, and a representative M. musculus sequence (the ~16.3 kb mitochondrial genome), at 100%, 100%, and 96.7% consensus pairwise identity, respectively; de novo assembly of the E. coli genome from raw reads yielded a single contig comprising 99.9% of the genome at 98.6% consensus pairwise identity. Simulated real-time analyses of in-flight sequence data using an automated bioinformatic pipeline and laptop-based genomic assembly demonstrated the feasibility of sequencing analysis and microbial identification aboard the ISS. These findings illustrate the potential for sequencing applications including disease diagnosis, environmental monitoring, and elucidating the molecular basis for how organisms respond to spaceflight.

Lanthanum-Based Metal-Organic Frameworks for Specific Detection of Sudan Virus RNA Conservative Sequences down to Single-Base Mismatch.

PubMed

Yang, Shui-Ping; Zhao, Wei; Hu, Pei-Pei; Wu, Ke-Yang; Jiang, Zhi-Hong; Bai, Li-Ping; Li, Min-Min; Chen, Jin-Xiang

2017-12-18

Reactions of La(NO 3 ) 3 ·6H 2 O with the polar, tritopic quaternized carboxylate ligands N-carboxymethyl-3,5-dicarboxylpyridinium bromide (H 3 CmdcpBr) and N-(4-carboxybenzyl)-3,5-dicarboxylpyridinium bromide (H 3 CbdcpBr) afford two water-stable metal-organic frameworks (MOFs) of {[La 4 (Cmdcp) 6 (H 2 O) 9 ]} n (1, 3D) and {[La 2 (Cbdcp) 3 (H 2 O) 10 ]} n (2, 2D). MOFs 1 and 2 absorb the carboxyfluorescein (FAM)-tagged probe DNA (P-DNA) and quench the fluorescence of FAM via a photoinduced electron transfer (PET) process. The nonemissive P-DNA@MOF hybrids thus formed in turn function as sensing platforms to distinguish conservative linear, single-stranded RNA sequences of Sudan virus with high selectivity and low detection limits of 112 and 67 pM, respectively (at a signal-to-noise ratio of 3). These hybrids also exhibit high specificity and discriminate down to single-base mismatch RNA sequences.

Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.

PubMed

Brunner, C; Hoffmann, K; Thiele, T; Schedler, U; Jehle, H; Resch-Genger, U

2015-04-01

Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a "green" dye and a "red" dye in a polymer matrix. These dyes present "Cy3" and "Cy5" analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.

Progress in ion torrent semiconductor chip based sequencing.

PubMed

Merriman, Barry; Rothberg, Jonathan M

2012-12-01

In order for next-generation sequencing to become widely used as a diagnostic in the healthcare industry, sequencing instrumentation will need to be mass produced with a high degree of quality and economy. One way to achieve this is to recast DNA sequencing in a format that fully leverages the manufacturing base created for computer chips, complementary metal-oxide semiconductor chip fabrication, which is the current pinnacle of large scale, high quality, low-cost manufacturing of high technology. To achieve this, ideally the entire sensory apparatus of the sequencer would be embodied in a standard semiconductor chip, manufactured in the same fab facilities used for logic and memory chips. Recently, such a sequencing chip, and the associated sequencing platform, has been developed and commercialized by Ion Torrent, a division of Life Technologies, Inc. Here we provide an overview of this semiconductor chip based sequencing technology, and summarize the progress made since its commercial introduction. We described in detail the progress in chip scaling, sequencing throughput, read length, and accuracy. We also summarize the enhancements in the associated platform, including sample preparation, data processing, and engagement of the broader development community through open source and crowdsourcing initiatives. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli

PubMed Central

Zampini, Massimiliano; Stevens, Pauline Rees; Pachebat, Justin A.; Kingston-Smith, Alison; Mur, Luis A. J.; Hayes, Finbarr

2015-01-01

The ability to assemble DNA sequences de novo through efficient and powerful DNA fabrication methods is one of the foundational technologies of synthetic biology. Gene synthesis, in particular, has been considered the main driver for the emergence of this new scientific discipline. Here we describe RapGene, a rapid gene assembly technique which was successfully tested for the synthesis and cloning of both prokaryotic and eukaryotic genes through a ligation independent approach. The method developed in this study is a complete bacterial gene synthesis platform for the quick, accurate and cost effective fabrication and cloning of gene-length sequences that employ the widely used host Escherichia coli. PMID:26062748

A dynamic bead-based microarray for parallel DNA detection

NASA Astrophysics Data System (ADS)

Sochol, R. D.; Casavant, B. P.; Dueck, M. E.; Lee, L. P.; Lin, L.

2011-05-01

A microfluidic system has been designed and constructed by means of micromachining processes to integrate both microfluidic mixing of mobile microbeads and hydrodynamic microbead arraying capabilities on a single chip to simultaneously detect multiple bio-molecules. The prototype system has four parallel reaction chambers, which include microchannels of 18 × 50 µm2 cross-sectional area and a microfluidic mixing section of 22 cm length. Parallel detection of multiple DNA oligonucleotide sequences was achieved via molecular beacon probes immobilized on polystyrene microbeads of 16 µm diameter. Experimental results show quantitative detection of three distinct DNA oligonucleotide sequences from the Hepatitis C viral (HCV) genome with single base-pair mismatch specificity. Our dynamic bead-based microarray offers an effective microfluidic platform to increase parallelization of reactions and improve microbead handling for various biological applications, including bio-molecule detection, medical diagnostics and drug screening.

A label-free ultrasensitive electrochemical DNA sensor based on thin-layer MoS2 nanosheets with high electrochemical activity.

PubMed

Wang, Xinxing; Nan, Fuxin; Zhao, Jinlong; Yang, Tao; Ge, Tong; Jiao, Kui

2015-02-15

A label-free and ultrasensitive electrochemical DNA biosensor, based on thin-layer molybdenum disulfide (MoS2) nanosheets sensing platform and differential pulse voltammetry detection, is constructed in this paper. The thin-layer MoS2 nanosheets were prepared via a simple ultrasound exfoliation method from bulk MoS2, which is simpler and no distortion compared with mechanical cleavage and lithium intercalation. Most importantly, this procedure allows the formation of MoS2 with enhanced electrochemical activity. Based on the high electrochemical activity and different affinity toward ssDNA versus dsDNA of the thin-layer MoS2 nanosheets sensing platform, the tlh gene sequence assay can be performed label-freely from 1.0 × 10(-16)M to 1.0 × 10(-10)M with a detection limit of 1.9 × 10(-17)M. Without labeling and the use of amplifiers, the detection method described here not only expands the application of MoS2, but also offers a viable alternative for DNA analysis, which has the priority in sensitivity, simplicity, and costs. Moreover, the proposed sensing platform has good electrocatalytic activity, and can be extended to detect more targets, such as guanine and adenine, which further expands the application of MoS2. Copyright © 2014 Elsevier B.V. All rights reserved.

Nanopore sensing of individual transcription factors bound to DNA

PubMed Central

Squires, Allison; Atas, Evrim; Meller, Amit

2015-01-01

Transcription factor (TF)-DNA interactions are the primary control point in regulation of gene expression. Characterization of these interactions is essential for understanding genetic regulation of biological systems and developing novel therapies to treat cellular malfunctions. Solid-state nanopores are a highly versatile class of single-molecule sensors that can provide rich information about local properties of long charged biopolymers using the current blockage patterns generated during analyte translocation, and provide a novel platform for characterization of TF-DNA interactions. The DNA-binding domain of the TF Early Growth Response Protein 1 (EGR1), a prototypical zinc finger protein known as zif268, is used as a model system for this study. zif268 adopts two distinct bound conformations corresponding to specific and nonspecific binding, according to the local DNA sequence. Here we implement a solid-state nanopore platform for direct, label- and tether-free single-molecule detection of zif268 bound to DNA. We demonstrate detection of single zif268 TFs bound to DNA according to current blockage sublevels and duration of translocation through the nanopore. We further show that the nanopore can detect and discriminate both specific and nonspecific binding conformations of zif268 on DNA via the distinct current blockage patterns corresponding to each of these two known binding modes. PMID:26109509

Nanopore sensing of individual transcription factors bound to DNA

NASA Astrophysics Data System (ADS)

Squires, Allison; Atas, Evrim; Meller, Amit

2015-06-01

Transcription factor (TF)-DNA interactions are the primary control point in regulation of gene expression. Characterization of these interactions is essential for understanding genetic regulation of biological systems and developing novel therapies to treat cellular malfunctions. Solid-state nanopores are a highly versatile class of single-molecule sensors that can provide rich information about local properties of long charged biopolymers using the current blockage patterns generated during analyte translocation, and provide a novel platform for characterization of TF-DNA interactions. The DNA-binding domain of the TF Early Growth Response Protein 1 (EGR1), a prototypical zinc finger protein known as zif268, is used as a model system for this study. zif268 adopts two distinct bound conformations corresponding to specific and nonspecific binding, according to the local DNA sequence. Here we implement a solid-state nanopore platform for direct, label- and tether-free single-molecule detection of zif268 bound to DNA. We demonstrate detection of single zif268 TFs bound to DNA according to current blockage sublevels and duration of translocation through the nanopore. We further show that the nanopore can detect and discriminate both specific and nonspecific binding conformations of zif268 on DNA via the distinct current blockage patterns corresponding to each of these two known binding modes.

A DNA 'barcode blitz': rapid digitization and sequencing of a natural history collection.

PubMed

Hebert, Paul D N; Dewaard, Jeremy R; Zakharov, Evgeny V; Prosser, Sean W J; Sones, Jayme E; McKeown, Jaclyn T A; Mantle, Beth; La Salle, John

2013-01-01

DNA barcoding protocols require the linkage of each sequence record to a voucher specimen that has, whenever possible, been authoritatively identified. Natural history collections would seem an ideal resource for barcode library construction, but they have never seen large-scale analysis because of concerns linked to DNA degradation. The present study examines the strength of this barrier, carrying out a comprehensive analysis of moth and butterfly (Lepidoptera) species in the Australian National Insect Collection. Protocols were developed that enabled tissue samples, specimen data, and images to be assembled rapidly. Using these methods, a five-person team processed 41,650 specimens representing 12,699 species in 14 weeks. Subsequent molecular analysis took about six months, reflecting the need for multiple rounds of PCR as sequence recovery was impacted by age, body size, and collection protocols. Despite these variables and the fact that specimens averaged 30.4 years old, barcode records were obtained from 86% of the species. In fact, one or more barcode compliant sequences (>487 bp) were recovered from virtually all species represented by five or more individuals, even when the youngest was 50 years old. By assembling specimen images, distributional data, and DNA barcode sequences on a web-accessible informatics platform, this study has greatly advanced accessibility to information on thousands of species. Moreover, much of the specimen data became publically accessible within days of its acquisition, while most sequence results saw release within three months. As such, this study reveals the speed with which DNA barcode workflows can mobilize biodiversity data, often providing the first web-accessible information for a species. These results further suggest that existing collections can enable the rapid development of a comprehensive DNA barcode library for the most diverse compartment of terrestrial biodiversity - insects.

Chimeric TALE recombinases with programmable DNA sequence specificity.

PubMed

Mercer, Andrew C; Gaj, Thomas; Fuller, Roberta P; Barbas, Carlos F

2012-11-01

Site-specific recombinases are powerful tools for genome engineering. Hyperactivated variants of the resolvase/invertase family of serine recombinases function without accessory factors, and thus can be re-targeted to sequences of interest by replacing native DNA-binding domains (DBDs) with engineered zinc-finger proteins (ZFPs). However, imperfect modularity with particular domains, lack of high-affinity binding to all DNA triplets, and difficulty in construction has hindered the widespread adoption of ZFPs in unspecialized laboratories. The discovery of a novel type of DBD in transcription activator-like effector (TALE) proteins from Xanthomonas provides an alternative to ZFPs. Here we describe chimeric TALE recombinases (TALERs): engineered fusions between a hyperactivated catalytic domain from the DNA invertase Gin and an optimized TALE architecture. We use a library of incrementally truncated TALE variants to identify TALER fusions that modify DNA with efficiency and specificity comparable to zinc-finger recombinases in bacterial cells. We also show that TALERs recombine DNA in mammalian cells. The TALER architecture described herein provides a platform for insertion of customized TALE domains, thus significantly expanding the targeting capacity of engineered recombinases and their potential applications in biotechnology and medicine.

High-throughput automated microfluidic sample preparation for accurate microbial genomics

PubMed Central

Kim, Soohong; De Jonghe, Joachim; Kulesa, Anthony B.; Feldman, David; Vatanen, Tommi; Bhattacharyya, Roby P.; Berdy, Brittany; Gomez, James; Nolan, Jill; Epstein, Slava; Blainey, Paul C.

2017-01-01

Low-cost shotgun DNA sequencing is transforming the microbial sciences. Sequencing instruments are so effective that sample preparation is now the key limiting factor. Here, we introduce a microfluidic sample preparation platform that integrates the key steps in cells to sequence library sample preparation for up to 96 samples and reduces DNA input requirements 100-fold while maintaining or improving data quality. The general-purpose microarchitecture we demonstrate supports workflows with arbitrary numbers of reaction and clean-up or capture steps. By reducing the sample quantity requirements, we enabled low-input (∼10,000 cells) whole-genome shotgun (WGS) sequencing of Mycobacterium tuberculosis and soil micro-colonies with superior results. We also leveraged the enhanced throughput to sequence ∼400 clinical Pseudomonas aeruginosa libraries and demonstrate excellent single-nucleotide polymorphism detection performance that explained phenotypically observed antibiotic resistance. Fully-integrated lab-on-chip sample preparation overcomes technical barriers to enable broader deployment of genomics across many basic research and translational applications. PMID:28128213

SPRi-based biosensing platforms for detection of specific DNA sequences using thiolate and dithiocarbamate assemblies

NASA Astrophysics Data System (ADS)

Drozd, Marcin; Pietrzak, Mariusz D.; Malinowska, Elżbieta

2018-05-01

The framework of presented study covers the development and examination of the analytical performance of surface plasmon resonance-based (SPR) DNA biosensors dedicated for a detection of model target oligonucleotide sequence. For this aim, various strategies of immobilization of DNA probes on gold transducers were tested. Besides the typical approaches: chemisorption of thiolated ssDNA (DNA-thiol) and physisorption of non-functionalized oligonucleotides, relatively new method based on chemisorption of dithiocarbamate-functionalized ssDNA (DNA-DTC) was applied for the first time for preparation of DNA-based SPR biosensor. The special emphasis was put on the correlation between the method of DNA immobilization and the composition of obtained receptor layer. The carried out studies focused on the examination of the capability of developed receptors layers to interact with both target DNA and DNA-functionalized AuNPs. It was found, that the detection limit of target DNA sequence (27 nb length) depends on the strategy of probe immobilization and backfilling method, and in the best case it amounted to 0,66 nM. Moreover, the application of ssDNA-functionalized gold nanoparticles (AuNPs) as plasmonic labels for secondary enhancement of SPR response is presented. The influence of spatial organization and surface density of a receptor layer on the ability to interact with DNA-functionalized AuNPs is discussed. Due to the best compatibility of receptors immobilized via DTC chemisorption: 1.47 ± 0.4 ·1012 molecules • cm-2 (with the calculated area occupied by single nanoparticle label of 132.7 nm2), DNA chemisorption based on DTCs is pointed as especially promising for DNA biosensors utilizing indirect detection in competitive assays.

SPRi-Based Biosensing Platforms for Detection of Specific DNA Sequences Using Thiolate and Dithiocarbamate Assemblies.

PubMed

Drozd, Marcin; Pietrzak, Mariusz D; Malinowska, Elżbieta

2018-01-01

The framework of presented study covers the development and examination of the analytical performance of surface plasmon resonance-based (SPR) DNA biosensors dedicated for a detection of model target oligonucleotide sequence. For this aim, various strategies of immobilization of DNA probes on gold transducers were tested. Besides the typical approaches: chemisorption of thiolated ssDNA (DNA-thiol) and physisorption of non-functionalized oligonucleotides, relatively new method based on chemisorption of dithiocarbamate-functionalized ssDNA (DNA-DTC) was applied for the first time for preparation of DNA-based SPR biosensor. The special emphasis was put on the correlation between the method of DNA immobilization and the composition of obtained receptor layer. The carried out studies focused on the examination of the capability of developed receptors layers to interact with both target DNA and DNA-functionalized AuNPs. It was found, that the detection limit of target DNA sequence (27 nb length) depends on the strategy of probe immobilization and backfilling method, and in the best case it amounted to 0.66 nM. Moreover, the application of ssDNA-functionalized gold nanoparticles (AuNPs) as plasmonic labels for secondary enhancement of SPR response is presented. The influence of spatial organization and surface density of a receptor layer on the ability to interact with DNA-functionalized AuNPs is discussed. Due to the best compatibility of receptors immobilized via DTC chemisorption: 1.47 ± 0.4 · 10 12 molecules · cm -2 (with the calculated area occupied by single nanoparticle label of ~132.7 nm 2 ), DNA chemisorption based on DTCs is pointed as especially promising for DNA biosensors utilizing indirect detection in competitive assays.

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization

PubMed Central

Willems, Hanny; Jacobs, An; Hadiwikarta, Wahyu Wijaya; Venken, Tom; Valkenborg, Dirk; Van Roy, Nadine; Vandesompele, Jo; Hooyberghs, Jef

2017-01-01

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine. PMID:28542229

Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs)

PubMed Central

2011-01-01

Background Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Results Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. Conclusions This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative environments, smart theranostic applications combining drug delivery with imaging of platform localization are within reach. The modular design of these USPIO nanoclusters enables future development of platforms for imaging and drug delivery targeted towards proteolytic activity in tumors and in advanced atherosclerotic plaques. PMID:21352596

Engineered CRISPR Systems for Next Generation Gene Therapies.

PubMed

Pineda, Michael; Moghadam, Farzaneh; Ebrahimkhani, Mo R; Kiani, Samira

2017-09-15

An ideal in vivo gene therapy platform provides safe, reprogrammable, and precise strategies which modulate cell and tissue gene regulatory networks with a high temporal and spatial resolution. Clustered regularly interspaced short palindromic repeats (CRISPR), a bacterial adoptive immune system, and its CRISPR-associated protein 9 (Cas9), have gained attention for the ability to target and modify DNA sequences on demand with unprecedented flexibility and precision. The precision and programmability of Cas9 is derived from its complexation with a guide-RNA (gRNA) that is complementary to a desired genomic sequence. CRISPR systems open-up widespread applications including genetic disease modeling, functional screens, and synthetic gene regulation. The plausibility of in vivo genetic engineering using CRISPR has garnered significant traction as a next generation in vivo therapeutic. However, there are hurdles that need to be addressed before CRISPR-based strategies are fully implemented. Some key issues center on the controllability of the CRISPR platform, including minimizing genomic-off target effects and maximizing in vivo gene editing efficiency, in vivo cellular delivery, and spatial-temporal regulation. The modifiable components of CRISPR systems: Cas9 protein, gRNA, delivery platform, and the form of CRISPR system delivered (DNA, RNA, or ribonucleoprotein) have recently been engineered independently to design a better genome engineering toolbox. This review focuses on evaluating CRISPR potential as a next generation in vivo gene therapy platform and discusses bioengineering advancements that can address challenges associated with clinical translation of this emerging technology.

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing.

PubMed

Nong, Rachel Yuan; Wu, Di; Yan, Junhong; Hammond, Maria; Gu, Gucci Jijuan; Kamali-Moghaddam, Masood; Landegren, Ulf; Darmanis, Spyros

2013-06-01

Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.

Massively Parallel, Molecular Analysis Platform Developed Using a CMOS Integrated Circuit With Biological Nanopores

PubMed Central

Roever, Stefan

2012-01-01

A massively parallel, low cost molecular analysis platform will dramatically change the nature of protein, molecular and genomics research, DNA sequencing, and ultimately, molecular diagnostics. An integrated circuit (IC) with 264 sensors was fabricated using standard CMOS semiconductor processing technology. Each of these sensors is individually controlled with precision analog circuitry and is capable of single molecule measurements. Under electronic and software control, the IC was used to demonstrate the feasibility of creating and detecting lipid bilayers and biological nanopores using wild type α-hemolysin. The ability to dynamically create bilayers over each of the sensors will greatly accelerate pore development and pore mutation analysis. In addition, the noise performance of the IC was measured to be 30fA(rms). With this noise performance, single base detection of DNA was demonstrated using α-hemolysin. The data shows that a single molecule, electrical detection platform using biological nanopores can be operationalized and can ultimately scale to millions of sensors. Such a massively parallel platform will revolutionize molecular analysis and will completely change the field of molecular diagnostics in the future.

Metabarcoding monitoring analysis: the pros and cons of using co-extracted environmental DNA and RNA data to assess offshore oil production impacts on benthic communities.

PubMed

Laroche, Olivier; Wood, Susanna A; Tremblay, Louis A; Lear, Gavin; Ellis, Joanne I; Pochon, Xavier

2017-01-01

Sequencing environmental DNA (eDNA) is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs) have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once) and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene) and eukaryotic (18S ribosomal RNA gene) eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand). Macro-infauna (visual classification of benthic invertebrates) and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs), by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from the oil production activity. Overall, the data appeared more robust when trimmed by shared OTUs, showing a greater effect of the platform on alpha- and beta-diversity. Trimming by shared OTUs likely removes taxa derived from legacy DNA and technical artefacts introduced through reverse transcriptase, polymerase-chain-reaction and sequencing. Findings from our scoping study suggest that metabarcoding-based biomonitoring surveys should, if funds, time and expertise allow, be assessed using both eDNA and eRNA products.

Metabarcoding monitoring analysis: the pros and cons of using co-extracted environmental DNA and RNA data to assess offshore oil production impacts on benthic communities

PubMed Central

Wood, Susanna A.; Tremblay, Louis A.; Lear, Gavin; Ellis, Joanne I.; Pochon, Xavier

2017-01-01

Sequencing environmental DNA (eDNA) is increasingly being used as an alternative to traditional morphological-based identification to characterize biological assemblages and monitor anthropogenic impacts in marine environments. Most studies only assess eDNA which, compared to eRNA, can persist longer in the environment after cell death. Therefore, eRNA may provide a more immediate census of the environment due to its relatively weaker stability, leading some researchers to advocate for the use of eRNA as an additional, or perhaps superior proxy for portraying ecological changes. A variety of pre-treatment techniques for screening eDNA and eRNA derived operational taxonomic units (OTUs) have been employed prior to statistical analyses, including removing singleton taxa (i.e., OTUs found only once) and discarding those not present in both eDNA and eRNA datasets. In this study, we used bacterial (16S ribosomal RNA gene) and eukaryotic (18S ribosomal RNA gene) eDNA- and eRNA-derived data from benthic communities collected at increasing distances along a transect from an oil production platform (Taranaki, New Zealand). Macro-infauna (visual classification of benthic invertebrates) and physico-chemical data were analyzed in parallel. We tested the effect of removing singleton taxa, and removing taxa not present in the eDNA and eRNA libraries from the same environmental sample (trimmed by shared OTUs), by comparing the impact of the oil production platform on alpha- and beta-diversity of the eDNA/eRNA-based biological assemblages, and by correlating these to the morphologically identified macro-faunal communities and the physico-chemical data. When trimmed by singletons, presence/absence information from eRNA data represented the best proxy to detect changes on species diversity for both bacteria and eukaryotes. However, assessment of quantitative beta-diversity from read abundance information of bacteria eRNA did not, contrary to eDNA, reveal any impact from the oil production activity. Overall, the data appeared more robust when trimmed by shared OTUs, showing a greater effect of the platform on alpha- and beta-diversity. Trimming by shared OTUs likely removes taxa derived from legacy DNA and technical artefacts introduced through reverse transcriptase, polymerase-chain-reaction and sequencing. Findings from our scoping study suggest that metabarcoding-based biomonitoring surveys should, if funds, time and expertise allow, be assessed using both eDNA and eRNA products. PMID:28533985

DIVA V2.0

DOE Office of Scientific and Technical Information (OSTI.GOV)

CHEN, JOANNA; SIMIRENKO, LISA; TAPASWI, MANJIRI

The DIVA software interfaces a process in which researchers design their DNA with a web-based graphical user interface, submit their designs to a central queue, and a few weeks later receive their sequence-verified clonal constructs. Each researcher independently designs the DNA to be constructed with a web-based BioCAD tool, and presses a button to submit their designs to a central queue. Researchers have web-based access to their DNA design queues, and can track the progress of their submitted designs as they progress from "evaluation", to "waiting for reagents", to "in progress", to "complete". Researchers access their completed constructs through themore » central DNA repository. Along the way, all DNA construction success/failure rates are captured in a central database. Once a design has been submitted to the queue, a small number of dedicated staff evaluate the design for feasibility and provide feedback to the responsible researcher if the design is either unreasonable (e.g., encompasses a combinatorial library of a billion constructs) or small design changes could significantly facilitate the downstream implementation process. The dedicated staff then use DNA assembly design automation software to optimize the DNA construction process for the design, leveraging existing parts from the DNA repository where possible and ordering synthetic DNA where necessary. SynTrack software manages the physical locations and availability of the various requisite reagents and process inputs (e.g., DNA templates). Once all requisite process inputs are available, the design progresses from "waiting for reagents" to "in progress" in the design queue. Human-readable and machine-parseable DNA construction protocols output by the DNA assembly design automation software are then executed by the dedicated staff exploiting lab automation devices wherever possible. Since the all employed DNA construction methods are sequence-agnostic, standardized (utilize the same enzymatic master mixes and reaction conditions), completely independent DNA construction tasks can be aggregated into the same multi-well plates and pursued in parallel. The resulting sets of cloned constructs can then be screened by high-throughput next-gen sequencing platforms for sequence correctness. A combination of long read-length (e.g., PacBio) and paired-end read platforms (e.g., Illumina) would be exploited depending the particular task at hand (e.g., PacBio might be sufficient to screen a set of pooled constructs with significant gene divergence). Post sequence verification, designs for which at least one correct clone was identified will progress to a "complete" status, while designs for which no correct clones wereidentified will progress to a "failure" status. Depending on the failure mode (e.g., no transformants), and how many prior attempts/variations of assembly protocol have been already made for a given design, subsequent attempts may be made or the design can progress to a "permanent failure" state. All success and failure rate information will be captured during the process, including at which stage a given clonal construction procedure failed (e.g., no PCR product) and what the exact failure was (e.g. assembly piece 2 missing). This success/failure rate data can be leveraged to refine the DNA assembly design process.« less

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening

PubMed Central

2017-01-01

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing. PMID:28199790

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.

PubMed

MacConnell, Andrew B; Price, Alexander K; Paegel, Brian M

2017-03-13

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.

RAD tag sequencing as a source of SNP markers in Cynara cardunculus L

PubMed Central

2012-01-01

Background The globe artichoke (Cynara cardunculus L. var. scolymus) genome is relatively poorly explored, especially compared to those of the other major Asteraceae crops sunflower and lettuce. No SNP markers are in the public domain. We have combined the recently developed restriction-site associated DNA (RAD) approach with the Illumina DNA sequencing platform to effect the rapid and mass discovery of SNP markers for C. cardunculus. Results RAD tags were sequenced from the genomic DNA of three C. cardunculus mapping population parents, generating 9.7 million reads, corresponding to ~1 Gbp of sequence. An assembly based on paired ends produced ~6.0 Mbp of genomic sequence, separated into ~19,000 contigs (mean length 312 bp), of which ~21% were fragments of putative coding sequence. The shared sequences allowed for the discovery of ~34,000 SNPs and nearly 800 indels, equivalent to a SNP frequency of 5.6 per 1,000 nt, and an indel frequency of 0.2 per 1,000 nt. A sample of heterozygous SNP loci was mapped by CAPS assays and this exercise provided validation of our mining criteria. The repetitive fraction of the genome had a high representation of retrotransposon sequence, followed by simple repeats, AT-low complexity regions and mobile DNA elements. The genomic k-mers distribution and CpG rate of C. cardunculus, compared with data derived from three whole genome-sequenced dicots species, provided a further evidence of the random representation of the C. cardunculus genome generated by RAD sampling. Conclusion The RAD tag sequencing approach is a cost-effective and rapid method to develop SNP markers in a highly heterozygous species. Our approach permitted to generate a large and robust SNP datasets by the adoption of optimized filtering criteria. PMID:22214349

Vidjil: A Web Platform for Analysis of High-Throughput Repertoire Sequencing.

PubMed

Duez, Marc; Giraud, Mathieu; Herbert, Ryan; Rocher, Tatiana; Salson, Mikaël; Thonier, Florian

2016-01-01

The B and T lymphocytes are white blood cells playing a key role in the adaptive immunity. A part of their DNA, called the V(D)J recombinations, is specific to each lymphocyte, and enables recognition of specific antigenes. Today, with new sequencing techniques, one can get billions of DNA sequences from these regions. With dedicated Repertoire Sequencing (RepSeq) methods, it is now possible to picture population of lymphocytes, and to monitor more accurately the immune response as well as pathologies such as leukemia. Vidjil is an open-source platform for the interactive analysis of high-throughput sequencing data from lymphocyte recombinations. It contains an algorithm gathering reads into clonotypes according to their V(D)J junctions, a web application made of a sample, experiment and patient database and a visualization for the analysis of clonotypes along the time. Vidjil is implemented in C++, Python and Javascript and licensed under the GPLv3 open-source license. Source code, binaries and a public web server are available at http://www.vidjil.org and at http://bioinfo.lille.inria.fr/vidjil. Using the Vidjil web application consists of four steps: 1. uploading a raw sequence file (typically a FASTQ); 2. running RepSeq analysis software; 3. visualizing the results; 4. annotating the results and saving them for future use. For the end-user, the Vidjil web application needs no specific installation and just requires a connection and a modern web browser. Vidjil is used by labs in hematology or immunology for research and clinical applications.

Vidjil: A Web Platform for Analysis of High-Throughput Repertoire Sequencing

PubMed Central

Duez, Marc; Herbert, Ryan; Rocher, Tatiana; Salson, Mikaël; Thonier, Florian

2016-01-01

Background The B and T lymphocytes are white blood cells playing a key role in the adaptive immunity. A part of their DNA, called the V(D)J recombinations, is specific to each lymphocyte, and enables recognition of specific antigenes. Today, with new sequencing techniques, one can get billions of DNA sequences from these regions. With dedicated Repertoire Sequencing (RepSeq) methods, it is now possible to picture population of lymphocytes, and to monitor more accurately the immune response as well as pathologies such as leukemia. Methods and Results Vidjil is an open-source platform for the interactive analysis of high-throughput sequencing data from lymphocyte recombinations. It contains an algorithm gathering reads into clonotypes according to their V(D)J junctions, a web application made of a sample, experiment and patient database and a visualization for the analysis of clonotypes along the time. Vidjil is implemented in C++, Python and Javascript and licensed under the GPLv3 open-source license. Source code, binaries and a public web server are available at http://www.vidjil.org and at http://bioinfo.lille.inria.fr/vidjil. Using the Vidjil web application consists of four steps: 1. uploading a raw sequence file (typically a FASTQ); 2. running RepSeq analysis software; 3. visualizing the results; 4. annotating the results and saving them for future use. For the end-user, the Vidjil web application needs no specific installation and just requires a connection and a modern web browser. Vidjil is used by labs in hematology or immunology for research and clinical applications. PMID:27835690

A Comparison Between Genotyping-by-sequencing and Array-based Scoring of SNPs for Genomic Prediction Accuracy in Winter Wheat

USDA-ARS?s Scientific Manuscript database

The utilization of DNA molecular markers in plant breeding to maximize selection response via marker assisted selection (MAS) and genomic selection (GS) has the potential to revolutionize plant breeding. A key factor affecting GS applicability is the choice of molecular marker platform. Genotypying-...

Detection of human microRNAs across miRNA Array and Next Generation DNA Sequencing Platforms

EPA Science Inventory

microRNA (miRNAs) are non-coding RNA molecules between 19 and 30 nucleotides in length that are believed to regulate approximately 30 per cent of all human genes. They act as negative regulators of their gene targets in many biological processes. Recent developments in microar...

Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

PubMed Central

Chatterjee, Aniruddha; Rodger, Euan J.; Stockwell, Peter A.; Weeks, Robert J.; Morison, Ian M.

2012-01-01

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background. PMID:23193365

An innovative platform for quick and flexible joining of assorted DNA fragments

DOE PAGES

De Paoli, Henrique Cestari; Tuskan, Gerald A.; Yang, Xiaohan

2016-01-13

Successful synthetic biology efforts rely on conceptual and experimental designs in combination with testing of multi-gene constructs. Despite recent progresses, several limitations still hinder the ability to flexibly assemble and collectively share different types of DNA segments. We describe an advanced system for joining DNA fragments from a universal library that automatically maintains open reading frames (ORFs) and does not require linkers, adaptors, sequence homology, amplification or mutation (domestication) of fragments in order to work properly. Moreover, we find that this system, which is enhanced by a unique buffer formulation, provides unforeseen capabilities for testing, and sharing, complex multi-gene circuitrymore » assembled from different DNA fragments.« less

Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling

NASA Astrophysics Data System (ADS)

Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I.

2017-02-01

In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.

A signal-amplified electrochemical DNA biosensor incorporated with a colorimetric internal control for Vibrio cholerae detection using shelf-ready reagents.

PubMed

Low, Kim-Fatt; Zain, Zainiharyati Mohd; Yean, Chan Yean

2017-01-15

A novel enzyme/nanoparticle-based DNA biosensing platform with dual colorimetric/electrochemical approach has been developed for the sequence-specific detection of the bacterium Vibrio cholerae, the causative agent of acute diarrheal disease in cholera. This assay platform exploits the use of shelf-stable and ready-to-use (shelf-ready) reagents to greatly simplify the bioanalysis procedures, allowing the assay platform to be more amenable to point-of-care applications. To assure maximum diagnosis reliability, an internal control (IC) capable of providing instant validation of results was incorporated into the assay. The microbial target, single-stranded DNA amplified with asymmetric PCR, was quantitatively detected via electrochemical stripping analysis of gold nanoparticle-loaded latex microspheres as a signal-amplified hybridization tag, while the incorporated IC was analyzed using a simplified horseradish peroxidase enzyme-based colorimetric scheme by simple visual observation of enzymatic color development. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 145 clinical isolate-spiked fecal specimens. The limits of detection were 0.5ng/ml of genomic DNA and 10 colony-forming units (CFU)/ml of bacterial cells with dynamic ranges of 0-100ng/ml (R 2 =0.992) and log 10 (1-10 4 CFU/ml) (R 2 =0.9918), respectively. An accelerated stability test revealed that the assay reagents were stable at temperatures of 4-37°C, with an estimated ambient shelf life of 200 days. The versatility of the biosensing platform makes it easily adaptable for quantitative detection of other microbial pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of the Ion PGM™ Hi-Q™ sequencing chemistry on sequence data quality.

PubMed

Churchill, Jennifer D; King, Jonathan L; Chakraborty, Ranajit; Budowle, Bruce

2016-09-01

Massively parallel sequencing (MPS) offers substantial improvements over current forensic DNA typing methodologies such as increased resolution, scalability, and throughput. The Ion PGM™ is a promising MPS platform for analysis of forensic biological evidence. The system employs a sequencing-by-synthesis chemistry on a semiconductor chip that measures a pH change due to the release of hydrogen ions as nucleotides are incorporated into the growing DNA strands. However, implementation of MPS into forensic laboratories requires a robust chemistry. Ion Torrent's Hi-Q™ Sequencing Chemistry was evaluated to determine if it could improve on the quality of the generated sequence data in association with selected genetic marker targets. The whole mitochondrial genome and the HID-Ion STR 10-plex panel were sequenced on the Ion PGM™ system with the Ion PGM™ Sequencing 400 Kit and the Ion PGM™ Hi-Q™ Sequencing Kit. Concordance, coverage, strand balance, noise, and deletion ratios were assessed in evaluating the performance of the Ion PGM™ Hi-Q™ Sequencing Kit. The results indicate that reliable, accurate data are generated and that sequencing through homopolymeric regions can be improved with the use of Ion Torrent's Hi-Q™ Sequencing Chemistry. Overall, the quality of the generated sequencing data supports the potential for use of the Ion PGM™ in forensic genetic laboratories.

The barley EST DNA Replication and Repair Database (bEST-DRRD) as a tool for the identification of the genes involved in DNA replication and repair.

PubMed

Gruszka, Damian; Marzec, Marek; Szarejko, Iwona

2012-06-14

The high level of conservation of genes that regulate DNA replication and repair indicates that they may serve as a source of information on the origin and evolution of the species and makes them a reliable system for the identification of cross-species homologs. Studies that had been conducted to date shed light on the processes of DNA replication and repair in bacteria, yeast and mammals. However, there is still much to be learned about the process of DNA damage repair in plants. These studies, which were conducted mainly using bioinformatics tools, enabled the list of genes that participate in various pathways of DNA repair in Arabidopsis thaliana (L.) Heynh to be outlined; however, information regarding these mechanisms in crop plants is still very limited. A similar, functional approach is particularly difficult for a species whose complete genomic sequences are still unavailable. One of the solutions is to apply ESTs (Expressed Sequence Tags) as the basis for gene identification. For the construction of the barley EST DNA Replication and Repair Database (bEST-DRRD), presented here, the Arabidopsis nucleotide and protein sequences involved in DNA replication and repair were used to browse for and retrieve the deposited sequences, derived from four barley (Hordeum vulgare L.) sequence databases, including the "Barley Genome version 0.05" database (encompassing ca. 90% of barley coding sequences) and from two databases covering the complete genomes of two monocot models: Oryza sativa L. and Brachypodium distachyon L. in order to identify homologous genes. Sequences of the categorised Arabidopsis queries are used for browsing the repositories, which are located on the ViroBLAST platform. The bEST-DRRD is currently used in our project during the identification and validation of the barley genes involved in DNA repair. The presented database provides information about the Arabidopsis genes involved in DNA replication and repair, their expression patterns and models of protein interactions. It was designed and established to provide an open-access tool for the identification of monocot homologs of known Arabidopsis genes that are responsible for DNA-related processes. The barley genes identified in the project are currently being analysed to validate their function.

GST-PRIME: an algorithm for genome-wide primer design.

PubMed

Leister, Dario; Varotto, Claudio

2007-01-01

The profiling of mRNA expression based on DNA arrays has become a powerful tool to study genome-wide transcription of genes in a number of organisms. GST-PRIME is a software package created to facilitate large-scale primer design for the amplification of probes to be immobilized on arrays for transcriptome analyses, even though it can be also applied in low-throughput approaches. GST-PRIME allows highly efficient, direct amplification of gene-sequence tags (GSTs) from genomic DNA (gDNA), starting from annotated genome or transcript sequences. GST-PRIME provides a customer-friendly platform for automatic primer design, and despite the relative simplicity of the algorithm, experimental tests in the model plant species Arabidopsis thaliana confirmed the reliability of the software. This chapter describes the algorithm used for primer design, its input and output files, and the installation of the standalone package and its use.

IM-TORNADO: a tool for comparison of 16S reads from paired-end libraries.

PubMed

Jeraldo, Patricio; Kalari, Krishna; Chen, Xianfeng; Bhavsar, Jaysheel; Mangalam, Ashutosh; White, Bryan; Nelson, Heidi; Kocher, Jean-Pierre; Chia, Nicholas

2014-01-01

16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads. We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity. IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq.

Millstone: software for multiplex microbial genome analysis and engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goodman, Daniel B.; Kuznetsov, Gleb; Lajoie, Marc J.

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. Here, we describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

Millstone: software for multiplex microbial genome analysis and engineering.

PubMed

Goodman, Daniel B; Kuznetsov, Gleb; Lajoie, Marc J; Ahern, Brian W; Napolitano, Michael G; Chen, Kevin Y; Chen, Changping; Church, George M

2017-05-25

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

Millstone: software for multiplex microbial genome analysis and engineering

DOE PAGES

Goodman, Daniel B.; Kuznetsov, Gleb; Lajoie, Marc J.; ...

2017-05-25

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. Here, we describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics

PubMed Central

Ardui, Simon; Ameur, Adam; Vermeesch, Joris R; Hestand, Matthew S

2018-01-01

Abstract Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio's single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing. PMID:29401301

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing.

PubMed

Zackay, Arie; Steinhoff, Christine

2010-12-15

Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org.

MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing

PubMed Central

2010-01-01

Background Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. Findings MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. Conclusions The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org. PMID:21159174

Multifluorophore DNA Origami Beacon as a Biosensing Platform.

PubMed

Selnihhin, Denis; Sparvath, Steffen Møller; Preus, Søren; Birkedal, Victoria; Andersen, Ebbe Sloth

2018-05-24

Biosensors play increasingly important roles in many fields, from clinical diagnosis to environmental monitoring, and there is a growing need for cheap and simple analytical devices. DNA nanotechnology provides methods for the creation of sophisticated biosensors, however many of the developed DNA-based sensors are limited by cumbersome and time-consuming readouts involving advanced experimental techniques. Here we describe design, construction, and characterization of an optical DNA origami nanobiosensor device exploiting arrays of precisely positioned organic fluorophores. Two arrays of donor and acceptor fluorophores make up a multifluorophore Förster resonance energy-transfer pair that results in a high-output signal for microscopic detection of single devices. Arrangement of fluorophores into arrays increases the signal-to-noise ratio, allowing detection of signal output from singular biosensors using a conventional fluorescence microscopy setup. Single device analysis enables detection of target DNA sequences in concentrations down to 100 pM in <45 min. We expect that the presented nanobiosensor can function as a general platform for incorporating sensor modules for a variety of targets and that the strong signal amplification properties may allow detection in portable microscope systems to be used for biosensor applications in the field.

Ultrasensitive and selective signal-on electrochemical DNA detection via exonuclease III catalysis and hybridization chain reaction amplification.

PubMed

Ren, Wang; Gao, Zhong Feng; Li, Nian Bing; Luo, Hong Qun

2015-01-15

This work reported a novel, ultrasensitive, and selective platform for electrochemical detection of DNA, employing an integration of exonuclease III (Exo-III) assisted target recycling and hybridization chain reaction (HCR) for the dual signal amplification strategy. The hairpin capture probe DNA (C-DNA) with an Exo-III 3' overhang end was self-assembled on a gold electrode. In the presence of target DNA (T-DNA), C-DNA hybridized with the T-DNA to form a duplex region, exposing its 5' complementary sequence (initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue, facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0 fM to 1.0 nM, and a detection limit as low as 0.2 fM. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma.

PubMed

Wrzeszczynski, Kazimierz O; Frank, Mayu O; Koyama, Takahiko; Rhrissorrakrai, Kahn; Robine, Nicolas; Utro, Filippo; Emde, Anne-Katrin; Chen, Bo-Juen; Arora, Kanika; Shah, Minita; Vacic, Vladimir; Norel, Raquel; Bilal, Erhan; Bergmann, Ewa A; Moore Vogel, Julia L; Bruce, Jeffrey N; Lassman, Andrew B; Canoll, Peter; Grommes, Christian; Harvey, Steve; Parida, Laxmi; Michelini, Vanessa V; Zody, Michael C; Jobanputra, Vaidehi; Royyuru, Ajay K; Darnell, Robert B

2017-08-01

To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. NCT02725684.

RPA binds histone H3-H4 and functions in DNA replication-coupled nucleosome assembly.

PubMed

Liu, Shaofeng; Xu, Zhiyun; Leng, He; Zheng, Pu; Yang, Jiayi; Chen, Kaifu; Feng, Jianxun; Li, Qing

2017-01-27

DNA replication-coupled nucleosome assembly is essential to maintain genome integrity and retain epigenetic information. Multiple involved histone chaperones have been identified, but how nucleosome assembly is coupled to DNA replication remains elusive. Here we show that replication protein A (RPA), an essential replisome component that binds single-stranded DNA, has a role in replication-coupled nucleosome assembly. RPA directly binds free H3-H4. Assays using a synthetic sequence that mimics freshly unwound single-stranded DNA at replication fork showed that RPA promotes DNA-(H3-H4) complex formation immediately adjacent to double-stranded DNA. Further, an RPA mutant defective in H3-H4 binding exhibited attenuated nucleosome assembly on nascent chromatin. Thus, we propose that RPA functions as a platform for targeting histone deposition to replication fork, through which RPA couples nucleosome assembly with ongoing DNA replication. Copyright © 2017, American Association for the Advancement of Science.

Characterization of NIST human mitochondrial DNA SRM-2392 and SRM-2392-I standard reference materials by next generation sequencing.

PubMed

Riman, Sarah; Kiesler, Kevin M; Borsuk, Lisa A; Vallone, Peter M

2017-07-01

Standard Reference Materials SRM 2392 and 2392-I are intended to provide quality control when amplifying and sequencing human mitochondrial genome sequences. The National Institute of Standards and Technology (NIST) offers these SRMs to laboratories performing DNA-based forensic human identification, molecular diagnosis of mitochondrial diseases, mutation detection, evolutionary anthropology, and genetic genealogy. The entire mtGenome (∼16569bp) of SRM 2392 and 2392-I have previously been characterized at NIST by Sanger sequencing. Herein, we used the sensitivity, specificity, and accuracy offered by next generation sequencing (NGS) to: (1) re-sequence the certified values of the SRM 2392 and 2392-I; (2) confirm Sanger data with a high coverage new sequencing technology; (3) detect lower level heteroplasmies (<20%); and thus (4) support mitochondrial sequencing communities in the adoption of NGS methods. To obtain a consensus sequence for the SRMs as well as identify and control any bias, sequencing was performed using two NGS platforms and data was analyzed using different bioinformatics pipelines. Our results confirm five low level heteroplasmy sites that were not previously observed with Sanger sequencing: three sites in the GM09947A template in SRM 2392 and two sites in the HL-60 template in SRM 2392-I. Copyright © 2017 Elsevier B.V. All rights reserved.

A microsphere-based assay for mutation analysis of the biotinidase gene using dried blood spots

PubMed Central

Lindau-Shepard, Barbara; Janik, David K.; Pass, Kenneth A.

2012-01-01

Biotinidase deficiency is an autosomal recessive syndrome caused by defects in the biotinidase gene, the product of which affects biotin metabolism. Newborn screening (NBS) for biotinidase deficiency can identify affected infants prior to onset of symptoms; biotin supplementation can resolve or prevent the clinical features. In NBS, dry blood spots (DBS) are usually tested for biotinidase enzyme activity by colorimetric analysis. By taking advantage of the multiplexing capabilities of the Luminex platform, we have developed a microsphere-based array genotyping method for the simultaneous detection of six disease causing mutations in the biotinidase gene, thereby permitting a second tier of molecular analysis. Genomic DNA was extracted from 3.2 mm DBS. Biotinidase gene sequences, containing the mutations of interest, were amplified by multiplexed polymerase chain reaction, followed by multiplexed allele-specific primer extension using universally tagged genotyping primers. The products were then hybridized to anti-tag carrying xTAG microspheres and detected on the Luminex platform. Genotypes were verified by sequencing. Genotyping results of 22 known biotinidase deficient samples by our xTAG biotinidase assay was in concordance with the results obtained from DNA sequencing, for all 6 mutations used in our panel. These results indicate that genotyping by an xTAG microsphere-based array is accurate, flexible, and can be adapted for high-throughput. Since NBS for biotinidase deficiency is by enzymatic assay, less than optimal quality of the DBS itself can compromise enzyme activity, while the DNA from these samples mostly remains unaffected. This assay warrants evaluation as a viable complement to the biotinidase semi-quantitative colorimetric assay. PMID:27625817

Single-molecule analysis of DNA cross-links using nanopore technology

NASA Astrophysics Data System (ADS)

Wolna, Anna H.

The alpha-hemolysin (alpha-HL) protein ion channel is a potential next-generation sequencing platform that has been extensively used to study nucleic acids at a single-molecule level. After applying a potential across a lipid bilayer, the imbedded alpha-HL allows monitoring of the duration and current levels of DNA translocation and immobilization. Because this method does not require DNA amplification prior to sequencing, all the DNA damage present in the cell at any given time will be present during the sequencing experiment. The goal of this research is to determine if these damage sites give distinguishable current levels beyond those observed for the canonical nucleobases. Because DNA cross-links are one of the most prevalent types of DNA damage occurring in vivo, the blockage current levels were determined for thymine-dimers, guanine(C8)-thymine(N3) cross-links and platinum adducts. All of these cross-links give a different blockage current level compared to the undamaged strands when immobilized in the ion channel, and they all can easily translocate across the alpha-HL channel. Additionally, the alpha-HL nanopore technique presents a unique opportunity to study the effects of DNA cross-links, such as thymine-dimers, on the secondary structure of DNA G-quadruplexes folded from the human telomere sequence. Using this single-molecule nanopore technique we can detect subtle structural differences that cannot be easily addressed using conventional methods. The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5'-TTAGGG human telomere sequence can fold into G-quadruplexes that adopt the hybrid fold in vivo. The telomere sequence is hypersensitive to UV-induced thymine-dimer (T=T) formation, and yet the presence of thymine dimers does not cause telomere shortening. The potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to understand how this damage is tolerated in telomeric DNA. The alpha-HL experiments determined that T=Ts disrupt double-chain reversal loop formation but are well tolerated in edgewise and diagonal loops of the hybrid G-quadruplexes. These studies demonstrated the power of the alpha-HL ion channel to analyze DNA modifications and secondary structures at a single-molecule level.

Ricebase: a breeding and genetics platform for rice, integrating individual molecular markers, pedigrees, and whole-genome-based data

USDA-ARS?s Scientific Manuscript database

Ricebase (http://ricebase.org) is an integrative genomic database for rice (Oryza sativa) with an emphasis on combining data sets in a way that maintains the key links between past and current genetic studies. Ricebase includes DNA sequence data, gene annotations, nucleotide variation data, and mol...

Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

PubMed

Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

2016-07-15

Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

SSR_pipeline--computer software for the identification of microsatellite sequences from paired-end Illumina high-throughput DNA sequence data

USGS Publications Warehouse

Miller, Mark P.; Knaus, Brian J.; Mullins, Thomas D.; Haig, Susan M.

2013-01-01

SSR_pipeline is a flexible set of programs designed to efficiently identify simple sequence repeats (SSRs; for example, microsatellites) from paired-end high-throughput Illumina DNA sequencing data. The program suite contains three analysis modules along with a fourth control module that can be used to automate analyses of large volumes of data. The modules are used to (1) identify the subset of paired-end sequences that pass quality standards, (2) align paired-end reads into a single composite DNA sequence, and (3) identify sequences that possess microsatellites conforming to user specified parameters. Each of the three separate analysis modules also can be used independently to provide greater flexibility or to work with FASTQ or FASTA files generated from other sequencing platforms (Roche 454, Ion Torrent, etc). All modules are implemented in the Python programming language and can therefore be used from nearly any computer operating system (Linux, Macintosh, Windows). The program suite relies on a compiled Python extension module to perform paired-end alignments. Instructions for compiling the extension from source code are provided in the documentation. Users who do not have Python installed on their computers or who do not have the ability to compile software also may choose to download packaged executable files. These files include all Python scripts, a copy of the compiled extension module, and a minimal installation of Python in a single binary executable. See program documentation for more information.

Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR

PubMed Central

Pinto, Fernando Lopes; Svensson, Håkan; Lindblad, Peter

2006-01-01

Background In order to overcome genomic DNA contamination in transcriptional studies, reverse template-specific polymerase chain reaction, a modification of reverse transcriptase polymerase chain reaction, is used. The possibility of using tags whose sequences are not found in the genome further improves reverse specific polymerase chain reaction experiments. Given the absence of software available to produce genome suitable tags, a simple tool to fulfill such need was developed. Results The program was developed in Perl, with separate use of the basic local alignment search tool, making the tool platform independent (known to run on Windows XP and Linux). In order to test the performance of the generated tags, several molecular experiments were performed. The results show that Tagenerator is capable of generating tags with good priming properties, which will deliberately not result in PCR amplification of genomic DNA. Conclusion The program Tagenerator is capable of generating tag sequences that combine genome absence with good priming properties for RT-PCR based experiments, circumventing the effects of genomic DNA contamination in an RNA sample. PMID:16820068

A comprehensive review on nano-molybdenum disulfide/DNA interfaces as emerging biosensing platforms.

PubMed

Kukkar, Manil; Mohanta, Girish C; Tuteja, Satish K; Kumar, Parveen; Bhadwal, Akhshay Singh; Samaddar, Pallabi; Kim, Ki-Hyun; Deep, Akash

2018-06-01

The development of nucleic acid-based portable platforms for the real-time analysis of diseases has attracted considerable scientific and commercial interest. Recently, 2D layered molybdenum sulfide (2D MoS 2 from here on) nanosheets have shown great potential for the development of next-generation platforms for efficient signal transduction. Through combination with DNA as a biorecognition medium, MoS 2 nanostructures have opened new opportunities to design and construct highly sensitive, specific, and commercially viable sensing devices. The use of specific short ssDNA sequences like aptamers has been proven to bind well with the unique transduction properties of 2D MoS 2 nanosheets to realize aptasensing devices. Such sensors can be operated on the principles of fluorescence, electro-cheumuluminescence, and electrochemistry with many advantageous features (e.g., robust biointerfacing through various conjugation chemistries, facile sensor assembly, high stability with regard to temperature/pH, and high affinity to target). This review encompasses the state of the art information on various design tactics and working principles of MoS 2 /DNA sensor technology which is emerging as one of the most sought-after and valuable fields with the advent of nucleic acid inspired devices. To help achieve a new milestone in biosensing applications, great potential of this emerging technique is described further with regard to sensitivity, specificity, operational convenience, and versatility. Copyright © 2018 Elsevier B.V. All rights reserved.

Microfluidic single-cell whole-transcriptome sequencing.

PubMed

Streets, Aaron M; Zhang, Xiannian; Cao, Chen; Pang, Yuhong; Wu, Xinglong; Xiong, Liang; Yang, Lu; Fu, Yusi; Zhao, Liang; Tang, Fuchou; Huang, Yanyi

2014-05-13

Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices.

PubMed

Linko, Veikko; Ora, Ari; Kostiainen, Mauri A

2015-10-01

DNA molecules can be assembled into custom predesigned shapes via hybridization of sequence-complementary domains. The folded structures have high spatial addressability and a tremendous potential to serve as platforms and active components in a plethora of bionanotechnological applications. DNA is a truly programmable material, and its nanoscale engineering thus opens up numerous attractive possibilities to develop novel methods for therapeutics. The tailored molecular devices could be used in targeting cells and triggering the cellular actions in the biological environment. In this review we focus on the DNA-based assemblies - primarily DNA origami nanostructures - that could perform complex tasks in cells and serve as smart drug-delivery vehicles in, for example, cancer therapy, prodrug medication, and enzyme replacement therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Designing Uniquely Addressable Bio-orthogonal Synthetic Scaffolds for DNA and RNA Origami.

PubMed

Kozyra, Jerzy; Ceccarelli, Alessandro; Torelli, Emanuela; Lopiccolo, Annunziata; Gu, Jing-Ying; Fellermann, Harold; Stimming, Ulrich; Krasnogor, Natalio

2017-07-21

Nanotechnology and synthetic biology are rapidly converging, with DNA origami being one of the leading bridging technologies. DNA origami was shown to work well in a wide array of biotic environments. However, the large majority of extant DNA origami scaffolds utilize bacteriophages or plasmid sequences thus severely limiting its future applicability as a bio-orthogonal nanotechnology platform. In this paper we present the design of biologically inert (i.e., "bio-orthogonal") origami scaffolds. The synthetic scaffolds have the additional advantage of being uniquely addressable (unlike biologically derived ones) and hence are better optimized for high-yield folding. We demonstrate our fully synthetic scaffold design with both DNA and RNA origamis and describe a protocol to produce these bio-orthogonal and uniquely addressable origami scaffolds.

Extracting DNA from FFPE Tissue Biospecimens Using User-Friendly Automated Technology: Is There an Impact on Yield or Quality?

PubMed

Mathieson, William; Guljar, Nafia; Sanchez, Ignacio; Sroya, Manveer; Thomas, Gerry A

2018-05-03

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue blocks is amenable to analytical techniques, including sequencing. DNA extraction protocols are typically long and complex, often involving an overnight proteinase K digest. Automated platforms that shorten and simplify the process are therefore an attractive proposition for users wanting a faster turn-around or to process large numbers of biospecimens. It is, however, unclear whether automated extraction systems return poorer DNA yields or quality than manual extractions performed by experienced technicians. We extracted DNA from 42 FFPE clinical tissue biospecimens using the QiaCube (Qiagen) and ExScale (ExScale Biospecimen Solutions) automated platforms, comparing DNA yields and integrities with those from manual extractions. The QIAamp DNA FFPE Spin Column Kit was used for manual and QiaCube DNA extractions and the ExScale extractions were performed using two of the manufacturer's magnetic bead kits: one extracting DNA only and the other simultaneously extracting DNA and RNA. In all automated extraction methods, DNA yields and integrities (assayed using DNA Integrity Numbers from a 4200 TapeStation and the qPCR-based Illumina FFPE QC Assay) were poorer than in the manual method, with the QiaCube system performing better than the ExScale system. However, ExScale was fastest, offered the highest reproducibility when extracting DNA only, and required the least intervention or technician experience. Thus, the extraction methods have different strengths and weaknesses, would appeal to different users with different requirements, and therefore, we cannot recommend one method over another.

Use of the melting curve assay as a means for high-throughput quantification of Illumina sequencing libraries.

PubMed

Shinozuka, Hiroshi; Forster, John W

2016-01-01

Background. Multiplexed sequencing is commonly performed on massively parallel short-read sequencing platforms such as Illumina, and the efficiency of library normalisation can affect the quality of the output dataset. Although several library normalisation approaches have been established, none are ideal for highly multiplexed sequencing due to issues of cost and/or processing time. Methods. An inexpensive and high-throughput library quantification method has been developed, based on an adaptation of the melting curve assay. Sequencing libraries were subjected to the assay using the Bio-Rad Laboratories CFX Connect(TM) Real-Time PCR Detection System. The library quantity was calculated through summation of reduction of relative fluorescence units between 86 and 95 °C. Results.PCR-enriched sequencing libraries are suitable for this quantification without pre-purification of DNA. Short DNA molecules, which ideally should be eliminated from the library for subsequent processing, were differentiated from the target DNA in a mixture on the basis of differences in melting temperature. Quantification results for long sequences targeted using the melting curve assay were correlated with those from existing methods (R (2) > 0.77), and that observed from MiSeq sequencing (R (2) = 0.82). Discussion.The results of multiplexed sequencing suggested that the normalisation performance of the described method is equivalent to that of another recently reported high-throughput bead-based method, BeNUS. However, costs for the melting curve assay are considerably lower and processing times shorter than those of other existing methods, suggesting greater suitability for highly multiplexed sequencing applications.

arrayCGHbase: an analysis platform for comparative genomic hybridization microarrays

PubMed Central

Menten, Björn; Pattyn, Filip; De Preter, Katleen; Robbrecht, Piet; Michels, Evi; Buysse, Karen; Mortier, Geert; De Paepe, Anne; van Vooren, Steven; Vermeesch, Joris; Moreau, Yves; De Moor, Bart; Vermeulen, Stefan; Speleman, Frank; Vandesompele, Jo

2005-01-01

Background The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH). One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment. Results We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment) supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser. Conclusion ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at . PMID:15910681

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing-moving toward barcoding the world.

PubMed

Liu, Shanlin; Yang, Chentao; Zhou, Chengran; Zhou, Xin

2017-12-01

Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)-based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn't show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes. © The Authors 2017. Published by Oxford University Press.

A base-modified PNA-graphene oxide platform as a turn-on fluorescence sensor for the detection of human telomeric repeats

NASA Astrophysics Data System (ADS)

Sabale, Pramod M.; George, Jerrin Thomas; Srivatsan, Seergazhi G.

2014-08-01

Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths. Our results demonstrate that this method, which does not involve a rigorous assay setup, would provide new opportunities to study G-quadruplex structures.Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths. Our results demonstrate that this method, which does not involve a rigorous assay setup, would provide new opportunities to study G-quadruplex structures. Electronic supplementary information (ESI) available. Figures, tables, experimental procedures and NMR spectra. See DOI: 10.1039/c4nr00878b

The iCRISPR platform for rapid genome editing in human pluripotent stem cells.

PubMed

Zhu, Zengrong; González, Federico; Huangfu, Danwei

2014-01-01

Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. Here, we first describe the establishment of the iCRISPR platform through TALEN-mediated targeting of inducible Cas9 expression cassettes into the AAVS1 locus. Next, we provide a series of technical procedures for using iCRISPR to achieve one-step knockout of one or multiple gene(s), "scarless" introduction of precise nucleotide alterations, as well as inducible knockout during hPSC differentiation. We present an optimized workflow, as well as guidelines for the selection of CRISPR targeting sequences and the design of single-stranded DNA (ssDNA) homology-directed DNA repair templates for the introduction of specific nucleotide alterations. We have successfully used these protocols in four different hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic modifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease.

Integrated electrochemical microsystems for genetic detection of pathogens at the point of care.

PubMed

Hsieh, Kuangwen; Ferguson, B Scott; Eisenstein, Michael; Plaxco, Kevin W; Soh, H Tom

2015-04-21

The capacity to achieve rapid, sensitive, specific, quantitative, and multiplexed genetic detection of pathogens via a robust, portable, point-of-care platform could transform many diagnostic applications. And while contemporary technologies have yet to effectively achieve this goal, the advent of microfluidics provides a potentially viable approach to this end by enabling the integration of sophisticated multistep biochemical assays (e.g., sample preparation, genetic amplification, and quantitative detection) in a monolithic, portable device from relatively small biological samples. Integrated electrochemical sensors offer a particularly promising solution to genetic detection because they do not require optical instrumentation and are readily compatible with both integrated circuit and microfluidic technologies. Nevertheless, the development of generalizable microfluidic electrochemical platforms that integrate sample preparation and amplification as well as quantitative and multiplexed detection remains a challenging and unsolved technical problem. Recognizing this unmet need, we have developed a series of microfluidic electrochemical DNA sensors that have progressively evolved to encompass each of these critical functionalities. For DNA detection, our platforms employ label-free, single-step, and sequence-specific electrochemical DNA (E-DNA) sensors, in which an electrode-bound, redox-reporter-modified DNA "probe" generates a current change after undergoing a hybridization-induced conformational change. After successfully integrating E-DNA sensors into a microfluidic chip format, we subsequently incorporated on-chip genetic amplification techniques including polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) to enable genetic detection at clinically relevant target concentrations. To maximize the potential point-of-care utility of our platforms, we have further integrated sample preparation via immunomagnetic separation, which allowed the detection of influenza virus directly from throat swabs and developed strategies for the multiplexed detection of related bacterial strains from the blood of septic mice. Finally, we developed an alternative electrochemical detection platform based on real-time LAMP, which not is only capable of detecting across a broad dynamic range of target concentrations, but also greatly simplifies quantitative measurement of nucleic acids. These efforts represent considerable progress toward the development of a true sample-in-answer-out platform for genetic detection of pathogens at the point of care. Given the many advantages of these systems, and the growing interest and innovative contributions from researchers in this field, we are optimistic that iterations of these systems will arrive in clinical settings in the foreseeable future.

BioWord: A sequence manipulation suite for Microsoft Word

PubMed Central

2012-01-01

Background The ability to manipulate, edit and process DNA and protein sequences has rapidly become a necessary skill for practicing biologists across a wide swath of disciplines. In spite of this, most everyday sequence manipulation tools are distributed across several programs and web servers, sometimes requiring installation and typically involving frequent switching between applications. To address this problem, here we have developed BioWord, a macro-enabled self-installing template for Microsoft Word documents that integrates an extensive suite of DNA and protein sequence manipulation tools. Results BioWord is distributed as a single macro-enabled template that self-installs with a single click. After installation, BioWord will open as a tab in the Office ribbon. Biologists can then easily manipulate DNA and protein sequences using a familiar interface and minimize the need to switch between applications. Beyond simple sequence manipulation, BioWord integrates functionality ranging from dyad search and consensus logos to motif discovery and pair-wise alignment. Written in Visual Basic for Applications (VBA) as an open source, object-oriented project, BioWord allows users with varying programming experience to expand and customize the program to better meet their own needs. Conclusions BioWord integrates a powerful set of tools for biological sequence manipulation within a handy, user-friendly tab in a widely used word processing software package. The use of a simple scripting language and an object-oriented scheme facilitates customization by users and provides a very accessible educational platform for introducing students to basic bioinformatics algorithms. PMID:22676326

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree

PubMed Central

2013-01-01

Background With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. Results The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. Conclusions Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology. PMID:23497206

BioWord: a sequence manipulation suite for Microsoft Word.

PubMed

Anzaldi, Laura J; Muñoz-Fernández, Daniel; Erill, Ivan

2012-06-07

The ability to manipulate, edit and process DNA and protein sequences has rapidly become a necessary skill for practicing biologists across a wide swath of disciplines. In spite of this, most everyday sequence manipulation tools are distributed across several programs and web servers, sometimes requiring installation and typically involving frequent switching between applications. To address this problem, here we have developed BioWord, a macro-enabled self-installing template for Microsoft Word documents that integrates an extensive suite of DNA and protein sequence manipulation tools. BioWord is distributed as a single macro-enabled template that self-installs with a single click. After installation, BioWord will open as a tab in the Office ribbon. Biologists can then easily manipulate DNA and protein sequences using a familiar interface and minimize the need to switch between applications. Beyond simple sequence manipulation, BioWord integrates functionality ranging from dyad search and consensus logos to motif discovery and pair-wise alignment. Written in Visual Basic for Applications (VBA) as an open source, object-oriented project, BioWord allows users with varying programming experience to expand and customize the program to better meet their own needs. BioWord integrates a powerful set of tools for biological sequence manipulation within a handy, user-friendly tab in a widely used word processing software package. The use of a simple scripting language and an object-oriented scheme facilitates customization by users and provides a very accessible educational platform for introducing students to basic bioinformatics algorithms.

Analysis of the whole mitochondrial genome: translation of the Ion Torrent Personal Genome Machine system to the diagnostic bench?

PubMed

Seneca, Sara; Vancampenhout, Kim; Van Coster, Rudy; Smet, Joél; Lissens, Willy; Vanlander, Arnaud; De Paepe, Boel; Jonckheere, An; Stouffs, Katrien; De Meirleir, Linda

2015-01-01

Next-generation sequencing (NGS), an innovative sequencing technology that enables the successful analysis of numerous gene sequences in a massive parallel sequencing approach, has revolutionized the field of molecular biology. Although NGS was introduced in a rather recent past, the technology has already demonstrated its potential and effectiveness in many research projects, and is now on the verge of being introduced into the diagnostic setting of routine laboratories to delineate the molecular basis of genetic disease in undiagnosed patient samples. We tested a benchtop device on retrospective genomic DNA (gDNA) samples of controls and patients with a clinical suspicion of a mitochondrial DNA disorder. This Ion Torrent Personal Genome Machine platform is a high-throughput sequencer with a fast turnaround time and reasonable running costs. We challenged the chemistry and technology with the analysis and processing of a mutational spectrum composed of samples with single-nucleotide substitutions, indels (insertions and deletions) and large single or multiple deletions, occasionally in heteroplasmy. The output data were compared with previously obtained conventional dideoxy sequencing results and the mitochondrial revised Cambridge Reference Sequence (rCRS). We were able to identify the majority of all nucleotide alterations, but three false-negative results were also encountered in the data set. At the same time, the poor performance of the PGM instrument in regions associated with homopolymeric stretches generated many false-positive miscalls demanding additional manual curation of the data.

Surface-Enhanced Raman Scattering Based Nonfluorescent Probe for Multiplex DNA Detection

PubMed Central

Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

2008-01-01

To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive and multiplex format, an alternative surface enhanced Raman scattering (SERS) based probe was designed and fabricated to covalently attach both DNA probing sequence and non-fluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the non-fluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA (ssDNA) to its complementary targets was successfully accomplished with a long-term goal to use non-fluorescent RTags in a Raman-based DNA microarray platform. PMID:17465531

DNA confinement in nanochannels: physics and biological applications

NASA Astrophysics Data System (ADS)

Reisner, Walter; Pedersen, Jonas N.; Austin, Robert H.

2012-10-01

DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement—including the effect of varying ionic strength—and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

DNA confinement in nanochannels: physics and biological applications.

PubMed

Reisner, Walter; Pedersen, Jonas N; Austin, Robert H

2012-10-01

DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement--including the effect of varying ionic strength--and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

JRC GMO-Matrix: a web application to support Genetically Modified Organisms detection strategies.

PubMed

Angers-Loustau, Alexandre; Petrillo, Mauro; Bonfini, Laura; Gatto, Francesco; Rosa, Sabrina; Patak, Alexandre; Kreysa, Joachim

2014-12-30

The polymerase chain reaction (PCR) is the current state of the art technique for DNA-based detection of Genetically Modified Organisms (GMOs). A typical control strategy starts by analyzing a sample for the presence of target sequences (GM-elements) known to be present in many GMOs. Positive findings from this "screening" are then confirmed with GM (event) specific test methods. A reliable knowledge of which GMOs are detected by combinations of GM-detection methods is thus crucial to minimize the verification efforts. In this article, we describe a novel platform that links the information of two unique databases built and maintained by the European Union Reference Laboratory for Genetically Modified Food and Feed (EU-RL GMFF) at the Joint Research Centre (JRC) of the European Commission, one containing the sequence information of known GM-events and the other validated PCR-based detection and identification methods. The new platform compiles in silico determinations of the detection of a wide range of GMOs by the available detection methods using existing scripts that simulate PCR amplification and, when present, probe binding. The correctness of the information has been verified by comparing the in silico conclusions to experimental results for a subset of forty-nine GM events and six methods. The JRC GMO-Matrix is unique for its reliance on DNA sequence data and its flexibility in integrating novel GMOs and new detection methods. Users can mine the database using a set of web interfaces that thus provide a valuable support to GMO control laboratories in planning and evaluating their GMO screening strategies. The platform is accessible at http://gmo-crl.jrc.ec.europa.eu/jrcgmomatrix/ .

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

PubMed Central

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta’ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-01-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology. PMID:27435636

[Current situation and prospect of breast cancer liquid biopsy].

PubMed

Zhou, B; Xin, L; Xu, L; Ye, J M; Liu, Y H

2018-02-01

Liquid biopsy is a diagnostic approach by analyzing body fluid samples. Peripheral blood is the most common sample. Urine, saliva, pleural effusion and ascites are also used. Now liquid biopsy is mainly used in the area of neoplasm diagnosis and treatment. Compared with traditional tissue biopsy, liquid biopsy is minimally invasive, convenient to sample and easy to repeat. Liquid biopsy mainly includes circulating tumor cells and circulating tumor DNA (ctDNA) detection. Detection of ctDNA requires sensitive and accurate methods. The progression of next-generation sequencing (NGS) and digital PCR promote the process of studies in ctDNA. In 2016, Nature published the result of whole-genome sequencing study of breast cancer. The study found 1 628 mutations of 93 protein-coding genes which may be driver mutations of breast cancer. The result of this study provided a new platform for breast cancer ctDNA studies. In recent years, there were many studies using ctDNA detection to monitor therapeutic effect and guide treatment. NGS is a promising technique in accessing genetic information and guiding targeted therapy. It must be emphasized that ctDNA detection using NGS is still at research stage. It is important to standardize ctDNA detection technique and perform prospective clinical researches. The time is not ripe for using ctDNA detection to guide large-scale breast cancer clinical practice at present.

Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

NASA Astrophysics Data System (ADS)

Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta'Ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

2016-07-01

The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology.

Research Associate | Center for Cancer Research

Cancer.gov

The Basic Science Program (BSP) at the Frederick National Laboratory for Cancer Research (FNLCR) pursues independent, multidisciplinary research programs in basic or applied molecular biology, immunology, retrovirology, cancer biology or human genetics. As part of the BSP, the Microbiome and Genetics Core (the Core) characterizes microbiomes by next-generation sequencing to determine their composition and variation, as influenced by immune, genetic, and host health factors. The Core provides support across a spectrum of processes, from nucleic acid isolation through bioinformatics and statistical analysis. KEY ROLES/RESPONSIBILITIES The Research Associate II will provide support in the areas of automated isolation, preparation, PCR and sequencing of DNA on next generation platforms (Illumina MiSeq and NextSeq). An opportunity exists to join the Core’s team of highly trained experimentalists and bioinformaticians working to characterize microbiome samples. The following represent requirements of the position: A minimum of five (5) years related of biomedical experience. Experience with high-throughput nucleic acid (DNA/RNA) extraction. Experience in performing PCR amplification (including quantitative real-time PCR). Experience or familiarity with robotic liquid handling protocols (especially on the Eppendorf epMotion 5073 or 5075 platforms). Experience in operating and maintaining benchtop Illumina sequencers (MiSeq and NextSeq). Ability to evaluate experimental quality and to troubleshoot molecular biology protocols. Experience with sample tracking, inventory management and biobanking. Ability to operate and communicate effectively in a team-oriented work environment.

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach

PubMed Central

Watson, Mick; Minot, Samuel S.; Rivera, Maria C.; Franklin, Rima B.

2017-01-01

Abstract Background: Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up to 98% assignment accuracy at the species level. The observed community proportions for “equal” and “rare” synthetic libraries were close to the known proportions, deviating from 0.1% to 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at <0.05% of DNA in the total mixture), 91% of reads were assigned to the correct species, 93% of reads were assigned to the correct genus, and >99% of reads were assigned to the correct family. Conclusions: At the current level of output and sequence quality (just under 4 × 103 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high-quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities. PMID:28327976

MinION™ nanopore sequencing of environmental metagenomes: a synthetic approach.

PubMed

Brown, Bonnie L; Watson, Mick; Minot, Samuel S; Rivera, Maria C; Franklin, Rima B

2017-03-01

Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up to 98% assignment accuracy at the species level. The observed community proportions for “equal” and “rare” synthetic libraries were close to the known proportions, deviating from 0.1% to 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at <0.05% of DNA in the total mixture), 91% of reads were assigned to the correct species, 93% of reads were assigned to the correct genus, and >99% of reads were assigned to the correct family. Conclusions: At the current level of output and sequence quality (just under 4 × 103 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high-quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities. © The Author 2017. Published by Oxford University Press.

Single nucleotide polymorphism (SNP) discovery in rainbow trout using restriction site associated DNA (RAD) sequencing of doubled haploids and assessment of polymorphism in a population survey

USDA-ARS?s Scientific Manuscript database

Background: Our goal is to produce a high-throughput SNP genotyping platform for genomic analyses in rainbow trout that will enable fine mapping of QTL, whole genome association studies, genomic selection for improved aquaculture production traits, and genetic analyses of wild populations that aid ...

Design and Development of a Technology Platform for DNA-Encoded Library Production and Affinity Selection.

PubMed

Castañón, Jesús; Román, José Pablo; Jessop, Theodore C; de Blas, Jesús; Haro, Rubén

2018-06-01

DNA-encoded libraries (DELs) have emerged as an efficient and cost-effective drug discovery tool for the exploration and screening of very large chemical space using small-molecule collections of unprecedented size. Herein, we report an integrated automation and informatics system designed to enhance the quality, efficiency, and throughput of the production and affinity selection of these libraries. The platform is governed by software developed according to a database-centric architecture to ensure data consistency, integrity, and availability. Through its versatile protocol management functionalities, this application captures the wide diversity of experimental processes involved with DEL technology, keeps track of working protocols in the database, and uses them to command robotic liquid handlers for the synthesis of libraries. This approach provides full traceability of building-blocks and DNA tags in each split-and-pool cycle. Affinity selection experiments and high-throughput sequencing reads are also captured in the database, and the results are automatically deconvoluted and visualized in customizable representations. Researchers can compare results of different experiments and use machine learning methods to discover patterns in data. As of this writing, the platform has been validated through the generation and affinity selection of various libraries, and it has become the cornerstone of the DEL production effort at Lilly.

NGS tools for traceability in candies as high processed food products: Ion Torrent PGM versus conventional PCR-cloning.

PubMed

Muñoz-Colmenero, Marta; Martínez, Jose Luis; Roca, Agustín; Garcia-Vazquez, Eva

2017-01-01

The Next Generation Sequencing methodologies are considered the next step within DNA-based methods and their applicability in different fields is being evaluated. Here, we tested the usefulness of the Ion Torrent Personal Genome Machine (PGM) in food traceability analyzing candies as a model of high processed foods, and compared the results with those obtained by PCR-cloning-sequencing (PCR-CS). The majority of samples exhibited consistency between methodologies, yielding more information and species per product from the PGM platform than PCR-CS. Significantly higher AT-content in sequences of the same species was also obtained from PGM. This together with some taxonomical discrepancies between methodologies suggest that the PGM platform is still pre-mature for its use in food traceability of complex highly processed products. It could be a good option for analysis of less complex food, saving time and cost per sample. Copyright © 2016 Elsevier Ltd. All rights reserved.

The complete chloroplast genome of Capsicum annuum var. glabriusculum using Illumina sequencing.

PubMed

Raveendar, Sebastin; Na, Young-Wang; Lee, Jung-Ro; Shim, Donghwan; Ma, Kyung-Ho; Lee, Sok-Young; Chung, Jong-Wook

2015-07-20

Chloroplast (cp) genome sequences provide a valuable source for DNA barcoding. Molecular phylogenetic studies have concentrated on DNA sequencing of conserved gene loci. However, this approach is time consuming and more difficult to implement when gene organization differs among species. Here we report the complete re-sequencing of the cp genome of Capsicum pepper (Capsicum annuum var. glabriusculum) using the Illumina platform. The total length of the cp genome is 156,817 bp with a 37.7% overall GC content. A pair of inverted repeats (IRs) of 50,284 bp were separated by a small single copy (SSC; 18,948 bp) and a large single copy (LSC; 87,446 bp). The number of cp genes in C. annuum var. glabriusculum is the same as that in other Capsicum species. Variations in the lengths of LSC; SSC and IR regions were the main contributors to the size variation in the cp genome of this species. A total of 125 simple sequence repeat (SSR) and 48 insertions or deletions variants were found by sequence alignment of Capsicum cp genome. These findings provide a foundation for further investigation of cp genome evolution in Capsicum and other higher plants.

IMG/VR: a database of cultured and uncultured DNA Viruses and retroviruses

DOE PAGES

Paez-Espino, David; Chen, I. -Min A.; Palaniappan, Krishna; ...

2016-10-30

Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from > 6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs aremore » grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparingwith external sequences, thus serving as an essential resource in the viral genomics community.« less

Genetic mutations in human rectal cancers detected by targeted sequencing.

PubMed

Bai, Jun; Gao, Jinglong; Mao, Zhijun; Wang, Jianhua; Li, Jianhui; Li, Wensheng; Lei, Yu; Li, Shuaishuai; Wu, Zhuo; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Lou, Feng; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Huang, Xue F; Chen, Si-Yi; Zhang, Enke

2015-10-01

Colorectal cancer (CRC) is widespread with significant mortality. Both inherited and sporadic mutations in various signaling pathways influence the development and progression of the cancer. Identifying genetic mutations in CRC is important for optimal patient treatment and many approaches currently exist to uncover these mutations, including next-generation sequencing (NGS) and commercially available kits. In the present study, we used a semiconductor-based targeted DNA-sequencing approach to sequence and identify genetic mutations in 91 human rectal cancer samples. Analysis revealed frequent mutations in KRAS (58.2%), TP53 (28.6%), APC (16.5%), FBXW7 (9.9%) and PIK3CA (9.9%), and additional mutations in BRAF, CTNNB1, ERBB2 and SMAD4 were also detected at lesser frequencies. Thirty-eight samples (41.8%) also contained two or more mutations, with common combination mutations occurring between KRAS and TP53 (42.1%), and KRAS and APC (31.6%). DNA sequencing for individual cancers is of clinical importance for targeted drug therapy and the advantages of such targeted gene sequencing over other NGS platforms or commercially available kits in sensitivity, cost and time effectiveness may aid clinicians in treating CRC patients in the near future.

IMG/VR: a database of cultured and uncultured DNA Viruses and retroviruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paez-Espino, David; Chen, I. -Min A.; Palaniappan, Krishna

Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from > 6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs aremore » grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparingwith external sequences, thus serving as an essential resource in the viral genomics community.« less

Next-generation sequencing: advances and applications in cancer diagnosis

PubMed Central

Serratì, Simona; De Summa, Simona; Pilato, Brunella; Petriella, Daniela; Lacalamita, Rosanna; Tommasi, Stefania; Pinto, Rosamaria

2016-01-01

Technological advances have led to the introduction of next-generation sequencing (NGS) platforms in cancer investigation. NGS allows massive parallel sequencing that affords maximal tumor genomic assessment. NGS approaches are different, and concern DNA and RNA analysis. DNA sequencing includes whole-genome, whole-exome, and targeted sequencing, which focuses on a selection of genes of interest for a specific disease. RNA sequencing facilitates the detection of alternative gene-spliced transcripts, posttranscriptional modifications, gene fusion, mutations/single-nucleotide polymorphisms, small and long noncoding RNAs, and changes in gene expression. Most applications are in the cancer research field, but lately NGS technology has been revolutionizing cancer molecular diagnostics, due to the many advantages it offers compared to traditional methods. There is greater knowledge on solid cancer diagnostics, and recent interest has been shown also in the field of hematologic cancer. In this review, we report the latest data on NGS diagnostic/predictive clinical applications in solid and hematologic cancers. Moreover, since the amount of NGS data produced is very large and their interpretation is very complex, we briefly discuss two bioinformatic aspects, variant-calling accuracy and copy-number variation detection, which are gaining a lot of importance in cancer-diagnostic assessment. PMID:27980425

Titanium Dioxide Nanoparticle-Based Interdigitated Electrodes: A Novel Current to Voltage DNA Biosensor Recognizes E. coli O157:H7.

PubMed

Nadzirah, Sh; Azizah, N; Hashim, Uda; Gopinath, Subash C B; Kashif, Mohd

2015-01-01

Nanoparticle-mediated bio-sensing promoted the development of novel sensors in the front of medical diagnosis. In the present study, we have generated and examined the potential of titanium dioxide (TiO2) crystalline nanoparticles with aluminium interdigitated electrode biosensor to specifically detect single-stranded E.coli O157:H7 DNA. The performance of this novel DNA biosensor was measured the electrical current response using a picoammeter. The sensor surface was chemically functionalized with (3-aminopropyl) triethoxysilane (APTES) to provide contact between the organic and inorganic surfaces of a single-stranded DNA probe and TiO2 nanoparticles while maintaining the sensing system's physical characteristics. The complement of the target DNA of E. coli O157:H7 to the carboxylate-probe DNA could be translated into electrical signals and confirmed by the increased conductivity in the current-to-voltage curves. The specificity experiments indicate that the biosensor can discriminate between the complementary sequences from the base-mismatched and the non-complementary sequences. After duplex formation, the complementary target sequence can be quantified over a wide range with a detection limit of 1.0 x 10(-13)M. With target DNA from the lysed E. coli O157:H7, we could attain similar sensitivity. Stability of DNA immobilized surface was calculated with the relative standard deviation (4.6%), displayed the retaining with 99% of its original response current until 6 months. This high-performance interdigitated DNA biosensor with high sensitivity, stability and non-fouling on a novel sensing platform is suitable for a wide range of biomolecular interactive analyses.

Titanium Dioxide Nanoparticle-Based Interdigitated Electrodes: A Novel Current to Voltage DNA Biosensor Recognizes E. coli O157:H7

PubMed Central

Nadzirah, Sh.; Azizah, N.; Hashim, Uda; Gopinath, Subash C. B.; Kashif, Mohd

2015-01-01

Nanoparticle-mediated bio-sensing promoted the development of novel sensors in the front of medical diagnosis. In the present study, we have generated and examined the potential of titanium dioxide (TiO2) crystalline nanoparticles with aluminium interdigitated electrode biosensor to specifically detect single-stranded E.coli O157:H7 DNA. The performance of this novel DNA biosensor was measured the electrical current response using a picoammeter. The sensor surface was chemically functionalized with (3-aminopropyl) triethoxysilane (APTES) to provide contact between the organic and inorganic surfaces of a single-stranded DNA probe and TiO2 nanoparticles while maintaining the sensing system’s physical characteristics. The complement of the target DNA of E. coli O157:H7 to the carboxylate-probe DNA could be translated into electrical signals and confirmed by the increased conductivity in the current-to-voltage curves. The specificity experiments indicate that the biosensor can discriminate between the complementary sequences from the base-mismatched and the non-complementary sequences. After duplex formation, the complementary target sequence can be quantified over a wide range with a detection limit of 1.0 x 10-13M. With target DNA from the lysed E. coli O157:H7, we could attain similar sensitivity. Stability of DNA immobilized surface was calculated with the relative standard deviation (4.6%), displayed the retaining with 99% of its original response current until 6 months. This high-performance interdigitated DNA biosensor with high sensitivity, stability and non-fouling on a novel sensing platform is suitable for a wide range of biomolecular interactive analyses. PMID:26445455

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes.

PubMed

Klymus, Katy E; Marshall, Nathaniel T; Stepien, Carol A

2017-01-01

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity.

Environmental DNA (eDNA) metabarcoding assays to detect invasive invertebrate species in the Great Lakes

PubMed Central

Klymus, Katy E.; Marshall, Nathaniel T.

2017-01-01

Describing and monitoring biodiversity comprise integral parts of ecosystem management. Recent research coupling metabarcoding and environmental DNA (eDNA) demonstrate that these methods can serve as important tools for surveying biodiversity, while significantly decreasing the time, expense and resources spent on traditional survey methods. The literature emphasizes the importance of genetic marker development, as the markers dictate the applicability, sensitivity and resolution ability of an eDNA assay. The present study developed two metabarcoding eDNA assays using the mtDNA 16S RNA gene with Illumina MiSeq platform to detect invertebrate fauna in the Laurentian Great Lakes and surrounding waterways, with a focus for use on invasive bivalve and gastropod species monitoring. We employed careful primer design and in vitro testing with mock communities to assess ability of the markers to amplify and sequence targeted species DNA, while retaining rank abundance information. In our mock communities, read abundances reflected the initial input abundance, with regressions having significant slopes (p<0.05) and high coefficients of determination (R2) for all comparisons. Tests on field environmental samples revealed similar ability of our markers to measure relative abundance. Due to the limited reference sequence data available for these invertebrate species, care must be taken when analyzing results and identifying sequence reads to species level. These markers extend eDNA metabarcoding research for molluscs and appear relevant to other invertebrate taxa, such as rotifers and bryozoans. Furthermore, the sphaeriid mussel assay is group-specific, exclusively amplifying bivalves in the Sphaeridae family and providing species-level identification. Our assays provide useful tools for managers and conservation scientists, facilitating early detection of invasive species as well as improving resolution of mollusc diversity. PMID:28542313

Adaptable gene-specific dye bias correction for two-channel DNA microarrays.

PubMed

Margaritis, Thanasis; Lijnzaad, Philip; van Leenen, Dik; Bouwmeester, Diane; Kemmeren, Patrick; van Hooff, Sander R; Holstege, Frank C P

2009-01-01

DNA microarray technology is a powerful tool for monitoring gene expression or for finding the location of DNA-bound proteins. DNA microarrays can suffer from gene-specific dye bias (GSDB), causing some probes to be affected more by the dye than by the sample. This results in large measurement errors, which vary considerably for different probes and also across different hybridizations. GSDB is not corrected by conventional normalization and has been difficult to address systematically because of its variance. We show that GSDB is influenced by label incorporation efficiency, explaining the variation of GSDB across different hybridizations. A correction method (Gene- And Slide-Specific Correction, GASSCO) is presented, whereby sequence-specific corrections are modulated by the overall bias of individual hybridizations. GASSCO outperforms earlier methods and works well on a variety of publically available datasets covering a range of platforms, organisms and applications, including ChIP on chip. A sequence-based model is also presented, which predicts which probes will suffer most from GSDB, useful for microarray probe design and correction of individual hybridizations. Software implementing the method is publicly available.

Adaptable gene-specific dye bias correction for two-channel DNA microarrays

PubMed Central

Margaritis, Thanasis; Lijnzaad, Philip; van Leenen, Dik; Bouwmeester, Diane; Kemmeren, Patrick; van Hooff, Sander R; Holstege, Frank CP

2009-01-01

DNA microarray technology is a powerful tool for monitoring gene expression or for finding the location of DNA-bound proteins. DNA microarrays can suffer from gene-specific dye bias (GSDB), causing some probes to be affected more by the dye than by the sample. This results in large measurement errors, which vary considerably for different probes and also across different hybridizations. GSDB is not corrected by conventional normalization and has been difficult to address systematically because of its variance. We show that GSDB is influenced by label incorporation efficiency, explaining the variation of GSDB across different hybridizations. A correction method (Gene- And Slide-Specific Correction, GASSCO) is presented, whereby sequence-specific corrections are modulated by the overall bias of individual hybridizations. GASSCO outperforms earlier methods and works well on a variety of publically available datasets covering a range of platforms, organisms and applications, including ChIP on chip. A sequence-based model is also presented, which predicts which probes will suffer most from GSDB, useful for microarray probe design and correction of individual hybridizations. Software implementing the method is publicly available. PMID:19401678

Nanostructured magnesium oxide biosensing platform for cholera detection

NASA Astrophysics Data System (ADS)

Patel, Manoj K.; Azahar Ali, Md.; Agrawal, Ved V.; Ansari, Z. A.; Ansari, S. G.; Malhotra, B. D.

2013-04-01

We report fabrication of highly crystalline nanostructured magnesium oxide (NanoMgO, size >30 nm) film electrophoretically deposited onto indium-tin-oxide (ITO) glass substrate for Vibrio cholerae detection. The single stranded deoxyribonucleic acid (ssDNA) probe, consisting of 23 bases (O1 gene sequence) immobilized onto NanoMgO/ITO electrode surface, has been characterized using electrochemical, Fourier Transform-Infra Red, and UltraViolet-visible spectroscopic techniques. The hybridization studies of ssDNA/NanoMgO/ITO bioelectrode with fragmented target DNA conducted using differential pulse voltammetry reveal sensitivity as 16.80 nA/ng/cm2, response time of 3 s, linearity as 100-500 ng/μL, and stability of about 120 days.

Surface-enhanced Raman scattering based nonfluorescent probe for multiplex DNA detection.

PubMed

Sun, Lan; Yu, Chenxu; Irudayaraj, Joseph

2007-06-01

To provide rapid and accurate detection of DNA markers in a straightforward, inexpensive, and multiplex format, an alternative surface-enhanced Raman scattering based probe was designed and fabricated to covalently attach both DNA probing sequence and nonfluorescent Raman tags to the surface of gold nanoparticles (DNA-AuP-RTag). The intensity of Raman signal of the probes could be controlled through the surface coverage of the nonfluorescent Raman tags (RTags). Detection sensitivity of these probes could be optimized by fine-tuning the amount of DNA molecules and RTags on the probes. Long-term stability of the DNA-AuP-RTag probes was found to be good (over 3 months). Excellent multiplexing capability of the DNA-AuP-RTag scheme was demonstrated by simultaneous identification of up to eight probes in a mixture. Detection of hybridization of single-stranded DNA to its complementary targets was successfully accomplished with a long-term goal to use nonfluorescent RTags in a Raman-based DNA microarray platform.

Next generation sequencing analysis reveals a relationship between rDNA unit diversity and locus number in Nicotiana diploids

PubMed Central

2012-01-01

Background Tandemly arranged nuclear ribosomal DNA (rDNA), encoding 18S, 5.8S and 26S ribosomal RNA (rRNA), exhibit concerted evolution, a pattern thought to result from the homogenisation of rDNA arrays. However rDNA homogeneity at the single nucleotide polymorphism (SNP) level has not been detailed in organisms with more than a few hundred copies of the rDNA unit. Here we study rDNA complexity in species with arrays consisting of thousands of units. Methods We examined homogeneity of genic (18S) and non-coding internally transcribed spacer (ITS1) regions of rDNA using Roche 454 and/or Illumina platforms in four angiosperm species, Nicotiana sylvestris, N. tomentosiformis, N. otophora and N. kawakamii. We compared the data with Southern blot hybridisation revealing the structure of intergenic spacer (IGS) sequences and with the number and distribution of rDNA loci. Results and Conclusions In all four species the intragenomic homogeneity of the 18S gene was high; a single ribotype makes up over 90% of the genes. However greater variation was observed in the ITS1 region, particularly in species with two or more rDNA loci, where >55% of rDNA units were a single ribotype, with the second most abundant variant accounted for >18% of units. IGS heterogeneity was high in all species. The increased number of ribotypes in ITS1 compared with 18S sequences may reflect rounds of incomplete homogenisation with strong selection for functional genic regions and relaxed selection on ITS1 variants. The relationship between the number of ITS1 ribotypes and the number of rDNA loci leads us to propose that rDNA evolution and complexity is influenced by locus number and/or amplification of orphaned rDNA units at new chromosomal locations. PMID:23259460

LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach.

PubMed

Fokkema, Ivo F A C; den Dunnen, Johan T; Taschner, Peter E M

2005-08-01

The completion of the human genome project has initiated, as well as provided the basis for, the collection and study of all sequence variation between individuals. Direct access to up-to-date information on sequence variation is currently provided most efficiently through web-based, gene-centered, locus-specific databases (LSDBs). We have developed the Leiden Open (source) Variation Database (LOVD) software approaching the "LSDB-in-a-Box" idea for the easy creation and maintenance of a fully web-based gene sequence variation database. LOVD is platform-independent and uses PHP and MySQL open source software only. The basic gene-centered and modular design of the database follows the recommendations of the Human Genome Variation Society (HGVS) and focuses on the collection and display of DNA sequence variations. With minimal effort, the LOVD platform is extendable with clinical data. The open set-up should both facilitate and promote functional extension with scripts written by the community. The LOVD software is freely available from the Leiden Muscular Dystrophy pages (www.DMD.nl/LOVD/). To promote the use of LOVD, we currently offer curators the possibility to set up an LSDB on our Leiden server. (c) 2005 Wiley-Liss, Inc.

CMSA: a heterogeneous CPU/GPU computing system for multiple similar RNA/DNA sequence alignment.

PubMed

Chen, Xi; Wang, Chen; Tang, Shanjiang; Yu, Ce; Zou, Quan

2017-06-24

The multiple sequence alignment (MSA) is a classic and powerful technique for sequence analysis in bioinformatics. With the rapid growth of biological datasets, MSA parallelization becomes necessary to keep its running time in an acceptable level. Although there are a lot of work on MSA problems, their approaches are either insufficient or contain some implicit assumptions that limit the generality of usage. First, the information of users' sequences, including the sizes of datasets and the lengths of sequences, can be of arbitrary values and are generally unknown before submitted, which are unfortunately ignored by previous work. Second, the center star strategy is suited for aligning similar sequences. But its first stage, center sequence selection, is highly time-consuming and requires further optimization. Moreover, given the heterogeneous CPU/GPU platform, prior studies consider the MSA parallelization on GPU devices only, making the CPUs idle during the computation. Co-run computation, however, can maximize the utilization of the computing resources by enabling the workload computation on both CPU and GPU simultaneously. This paper presents CMSA, a robust and efficient MSA system for large-scale datasets on the heterogeneous CPU/GPU platform. It performs and optimizes multiple sequence alignment automatically for users' submitted sequences without any assumptions. CMSA adopts the co-run computation model so that both CPU and GPU devices are fully utilized. Moreover, CMSA proposes an improved center star strategy that reduces the time complexity of its center sequence selection process from O(mn 2 ) to O(mn). The experimental results show that CMSA achieves an up to 11× speedup and outperforms the state-of-the-art software. CMSA focuses on the multiple similar RNA/DNA sequence alignment and proposes a novel bitmap based algorithm to improve the center star strategy. We can conclude that harvesting the high performance of modern GPU is a promising approach to accelerate multiple sequence alignment. Besides, adopting the co-run computation model can maximize the entire system utilization significantly. The source code is available at https://github.com/wangvsa/CMSA .

Structural basis of DNA sequence recognition by the response regulator PhoP in Mycobacterium tuberculosis.

PubMed

He, Xiaoyuan; Wang, Liqin; Wang, Shuishu

2016-04-15

The transcriptional regulator PhoP is an essential virulence factor in Mycobacterium tuberculosis, and it presents a target for the development of new anti-tuberculosis drugs and attenuated tuberculosis vaccine strains. PhoP binds to DNA as a highly cooperative dimer by recognizing direct repeats of 7-bp motifs with a 4-bp spacer. To elucidate the PhoP-DNA binding mechanism, we determined the crystal structure of the PhoP-DNA complex. The structure revealed a tandem PhoP dimer that bound to the direct repeat. The surprising tandem arrangement of the receiver domains allowed the four domains of the PhoP dimer to form a compact structure, accounting for the strict requirement of a 4-bp spacer and the highly cooperative binding of the dimer. The PhoP-DNA interactions exclusively involved the effector domain. The sequence-recognition helix made contact with the bases of the 7-bp motif in the major groove, and the wing interacted with the adjacent minor groove. The structure provides a starting point for the elucidation of the mechanism by which PhoP regulates the virulence of M. tuberculosis and guides the design of screening platforms for PhoP inhibitors.

FBIS: A regional DNA barcode archival & analysis system for Indian fishes.

PubMed

Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

2012-01-01

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. The database is available for free at http://mail.nbfgr.res.in/fbis/

DNA-stabilized silver nanoclusters and carbon nanoparticles oxide: A sensitive platform for label-free fluorescence turn-on detection of HIV-DNA sequences.

PubMed

Ye, Yu-Dan; Xia, Li; Xu, Dang-Dang; Xing, Xiao-Jing; Pang, Dai-Wen; Tang, Hong-Wu

2016-11-15

Based on the remarkable difference between the interactions of carbon nanoparticles (CNPs) oxide with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), and the fact that fluorescence of DNA-stabilized silver nanoclusters (AgNCs) can be quenched by CNPs oxide, DNA-functionalized AgNCs were applied as label-free fluorescence probes and a novel fluorescence resonance energy transfer (FRET) sensor was successfully constructed for the detection of human immunodeficiency virus (HIV) DNA sequences. CNPs oxide were prepared with the oxidation of candle soot, hence it is simple, time-saving and low-cost. The strategy of dual AgNCs probes was applied to improve the detection sensitivity by using dual- probe capturing the same target DNA in a sandwich mode and as the fluorescence donor, and using CNPs oxide as the acceptor. In the presence of target DNA, a dsDNA hybrid forms, leading to the desorption of the ssDNA-AgNCs probes from CNPs oxide, and the recovering of fluorescence of the AgNCs in a HIV-DNA concentration-dependent manner. The results show that HIV-DNA can be detected in the range of 1-50nM with a detection limit of 0.40nM in aqueous buffer. The method is simple, rapid and sensitive with no need of labeled fluorescent probes, and moreover, the design of fluorescent dual-probe makes full use of the excellent fluorescence property of AgNCs and further improves the detection sensitivity. Copyright © 2016 Elsevier B.V. All rights reserved.

EST Express: PHP/MySQL based automated annotation of ESTs from expression libraries

PubMed Central

Smith, Robin P; Buchser, William J; Lemmon, Marcus B; Pardinas, Jose R; Bixby, John L; Lemmon, Vance P

2008-01-01

Background Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds. Results We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples) produced by subtractive hybridization. Conclusion EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects. PMID:18402700

EST Express: PHP/MySQL based automated annotation of ESTs from expression libraries.

PubMed

Smith, Robin P; Buchser, William J; Lemmon, Marcus B; Pardinas, Jose R; Bixby, John L; Lemmon, Vance P

2008-04-10

Several biological techniques result in the acquisition of functional sets of cDNAs that must be sequenced and analyzed. The emergence of redundant databases such as UniGene and centralized annotation engines such as Entrez Gene has allowed the development of software that can analyze a great number of sequences in a matter of seconds. We have developed "EST Express", a suite of analytical tools that identify and annotate ESTs originating from specific mRNA populations. The software consists of a user-friendly GUI powered by PHP and MySQL that allows for online collaboration between researchers and continuity with UniGene, Entrez Gene and RefSeq. Two key features of the software include a novel, simplified Entrez Gene parser and tools to manage cDNA library sequencing projects. We have tested the software on a large data set (2,016 samples) produced by subtractive hybridization. EST Express is an open-source, cross-platform web server application that imports sequences from cDNA libraries, such as those generated through subtractive hybridization or yeast two-hybrid screens. It then provides several layers of annotation based on Entrez Gene and RefSeq to allow the user to highlight useful genes and manage cDNA library projects.

Genome-wide mapping of DNase I hypersensitive sites in rare cell populations using single-cell DNase sequencing.

PubMed

Cooper, James; Ding, Yi; Song, Jiuzhou; Zhao, Keji

2017-11-01

Increased chromatin accessibility is a feature of cell-type-specific cis-regulatory elements; therefore, mapping of DNase I hypersensitive sites (DHSs) enables the detection of active regulatory elements of transcription, including promoters, enhancers, insulators and locus-control regions. Single-cell DNase sequencing (scDNase-seq) is a method of detecting genome-wide DHSs when starting with either single cells or <1,000 cells from primary cell sources. This technique enables genome-wide mapping of hypersensitive sites in a wide range of cell populations that cannot be analyzed using conventional DNase I sequencing because of the requirement for millions of starting cells. Fresh cells, formaldehyde-cross-linked cells or cells recovered from formalin-fixed paraffin-embedded (FFPE) tissue slides are suitable for scDNase-seq assays. To generate scDNase-seq libraries, cells are lysed and then digested with DNase I. Circular carrier plasmid DNA is included during subsequent DNA purification and library preparation steps to prevent loss of the small quantity of DHS DNA. Libraries are generated for high-throughput sequencing on the Illumina platform using standard methods. Preparation of scDNase-seq libraries requires only 2 d. The materials and molecular biology techniques described in this protocol should be accessible to any general molecular biology laboratory. Processing of high-throughput sequencing data requires basic bioinformatics skills and uses publicly available bioinformatics software.

IM-TORNADO: A Tool for Comparison of 16S Reads from Paired-End Libraries

PubMed Central

Jeraldo, Patricio; Kalari, Krishna; Chen, Xianfeng; Bhavsar, Jaysheel; Mangalam, Ashutosh; White, Bryan; Nelson, Heidi; Kocher, Jean-Pierre; Chia, Nicholas

2014-01-01

Motivation 16S rDNA hypervariable tag sequencing has become the de facto method for accessing microbial diversity. Illumina paired-end sequencing, which produces two separate reads for each DNA fragment, has become the platform of choice for this application. However, when the two reads do not overlap, existing computational pipelines analyze data from read separately and underutilize the information contained in the paired-end reads. Results We created a workflow known as Illinois Mayo Taxon Organization from RNA Dataset Operations (IM-TORNADO) for processing non-overlapping reads while retaining maximal information content. Using synthetic mock datasets, we show that the use of both reads produced answers with greater correlation to those from full length 16S rDNA when looking at taxonomy, phylogeny, and beta-diversity. Availability and Implementation IM-TORNADO is freely available at http://sourceforge.net/projects/imtornado and produces BIOM format output for cross compatibility with other pipelines such as QIIME, mothur, and phyloseq. PMID:25506826

Development and characterization of a microheater array device for real-time DNA mutation detection

NASA Astrophysics Data System (ADS)

Williams, Layne; Okandan, Murat; Chagovetz, Alex; Blair, Steve

2008-04-01

DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tm's in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described.

Development and characterization of a microheater array device for real-time DNA mutation detection

NASA Astrophysics Data System (ADS)

Williams, Layne; Okandan, Murat; Chagovetz, Alex; Blair, Steve

2008-02-01

DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tm's in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described.

Label-free electrical detection of pyrophosphate generated from DNA polymerase reactions on field-effect devices.

PubMed

Credo, Grace M; Su, Xing; Wu, Kai; Elibol, Oguz H; Liu, David J; Reddy, Bobby; Tsai, Ta-Wei; Dorvel, Brian R; Daniels, Jonathan S; Bashir, Rashid; Varma, Madoo

2012-03-21

We introduce a label-free approach for sensing polymerase reactions on deoxyribonucleic acid (DNA) using a chelator-modified silicon-on-insulator field-effect transistor (SOI-FET) that exhibits selective and reversible electrical response to pyrophosphate anions. The chemical modification of the sensor surface was designed to include rolling-circle amplification (RCA) DNA colonies for locally enhanced pyrophosphate (PPi) signal generation and sensors with immobilized chelators for capture and surface-sensitive detection of diffusible reaction by-products. While detecting arrays of enzymatic base incorporation reactions is typically accomplished using optical fluorescence or chemiluminescence techniques, our results suggest that it is possible to develop scalable and portable PPi-specific sensors and platforms for broad biomedical applications such as DNA sequencing and microbe detection using surface-sensitive electrical readout techniques.

Filling reference gaps via assembling DNA barcodes using high-throughput sequencing—moving toward barcoding the world

PubMed Central

Zhou, Chengran

2017-01-01

Abstract Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)–based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn’t show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes. PMID:29077841

High-throughput sequencing of complete human mtDNA genomes from the Caucasus and West Asia: high diversity and demographic inferences.

PubMed

Schönberg, Anna; Theunert, Christoph; Li, Mingkun; Stoneking, Mark; Nasidze, Ivan

2011-09-01

To investigate the demographic history of human populations from the Caucasus and surrounding regions, we used high-throughput sequencing to generate 147 complete mtDNA genome sequences from random samples of individuals from three groups from the Caucasus (Armenians, Azeri and Georgians), and one group each from Iran and Turkey. Overall diversity is very high, with 144 different sequences that fall into 97 different haplogroups found among the 147 individuals. Bayesian skyline plots (BSPs) of population size change through time show a population expansion around 40-50 kya, followed by a constant population size, and then another expansion around 15-18 kya for the groups from the Caucasus and Iran. The BSP for Turkey differs the most from the others, with an increase from 35 to 50 kya followed by a prolonged period of constant population size, and no indication of a second period of growth. An approximate Bayesian computation approach was used to estimate divergence times between each pair of populations; the oldest divergence times were between Turkey and the other four groups from the South Caucasus and Iran (~400-600 generations), while the divergence time of the three Caucasus groups from each other was comparable to their divergence time from Iran (average of ~360 generations). These results illustrate the value of random sampling of complete mtDNA genome sequences that can be obtained with high-throughput sequencing platforms.

Highly multiplexed subcellular RNA sequencing in situ

PubMed Central

Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Yang, Joyce L.; Terry, Richard; Jeanty, Sauveur S. F.; Li, Chao; Amamoto, Ryoji; Peters, Derek T.; Turczyk, Brian M.; Marblestone, Adam H.; Inverso, Samuel A.; Bernard, Amy; Mali, Prashant; Rios, Xavier; Aach, John; Church, George M.

2014-01-01

Understanding the spatial organization of gene expression with single nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8,742 genes in situ, we examined RNA expression and localization in human primary fibroblasts using a simulated wound healing assay. FISSEQ is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ. PMID:24578530

A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis

PubMed Central

Down, Thomas A.; Rakyan, Vardhman K.; Turner, Daniel J.; Flicek, Paul; Li, Heng; Kulesha, Eugene; Gräf, Stefan; Johnson, Nathan; Herrero, Javier; Tomazou, Eleni M.; Thorne, Natalie P.; Bäckdahl, Liselotte; Herberth, Marlis; Howe, Kevin L.; Jackson, David K.; Miretti, Marcos M.; Marioni, John C.; Birney, Ewan; Hubbard, Tim J. P.; Durbin, Richard; Tavaré, Simon; Beck, Stephan

2009-01-01

DNA methylation is an indispensible epigenetic modification of mammalian genomes. Consequently there is great interest in strategies for genome-wide/whole-genome DNA methylation analysis, and immunoprecipitation-based methods have proven to be a powerful option. Such methods are rapidly shifting the bottleneck from data generation to data analysis, necessitating the development of better analytical tools. Until now, a major analytical difficulty associated with immunoprecipitation-based DNA methylation profiling has been the inability to estimate absolute methylation levels. Here we report the development of a novel cross-platform algorithm – Bayesian Tool for Methylation Analysis (Batman) – for analyzing Methylated DNA Immunoprecipitation (MeDIP) profiles generated using arrays (MeDIP-chip) or next-generation sequencing (MeDIP-seq). The latter is an approach we have developed to elucidate the first high-resolution whole-genome DNA methylation profile (DNA methylome) of any mammalian genome. MeDIP-seq/MeDIP-chip combined with Batman represent robust, quantitative, and cost-effective functional genomic strategies for elucidating the function of DNA methylation. PMID:18612301

Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi.

PubMed

Solomon, Kevin V; Henske, John K; Theodorou, Michael K; O'Malley, Michelle A

2016-04-01

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at -80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long with negligible growth impact. Genomic DNA extraction for these isolates was optimized to yield samples compatible with next generation sequencing platforms (e.g. Illumina, PacBio). Popular DNA isolation kits and precipitation protocols yielded preps that were unsuitable for sequencing due to carbohydrate contaminants from the chitin-rich cell wall and extensive energy reserves of gut fungi. To address this, we identified a proprietary method optimized for hardy plant samples that rapidly yielded DNA fragments in excess of 10 kb with minimal RNA, protein or carbohydrate contamination. Collectively, these techniques serve as fundamental tools to manipulate powerful biomass-degrading gut fungi and improve their accessibility among researchers. Copyright © 2015 Elsevier Ltd. All rights reserved.

Label-free screening of single biomolecules through resistive pulse sensing technology for precision medicine applications

NASA Astrophysics Data System (ADS)

Harrer, S.; Kim, S. C.; Schieber, C.; Kannam, S.; Gunn, N.; Moore, S.; Scott, D.; Bathgate, R.; Skafidas, S.; Wagner, J. M.

2015-05-01

Employing integrated nano- and microfluidic circuits for detecting and characterizing biological compounds through resistive pulse sensing technology is a vibrant area of research at the interface of biotechnology and nanotechnology. Resistive pulse sensing platforms can be customized to study virtually any particle of choice which can be threaded through a fluidic channel and enable label-free single-particle interrogation with the primary read-out signal being an electric current fingerprint. The ability to perform label-free molecular screening with single-molecule and even single binding site resolution makes resistive pulse sensing technology a powerful tool for analyzing the smallest units of biological systems and how they interact with each other on a molecular level. This task is at the core of experimental systems biology and in particular ‘omics research which in combination with next-generation DNA-sequencing and next-generation drug discovery and design forms the foundation of a novel disruptive medical paradigm commonly referred to as personalized medicine or precision medicine. DNA-sequencing has approached the 1000-Dollar-Genome milestone allowing for decoding a complete human genome with unmatched speed and at low cost. Increased sequencing efficiency yields massive amounts of genomic data. Analyzing this data in combination with medical and biometric health data eventually enables understanding the pathways from individual genes to physiological functions. Access to this information triggers fundamental questions for doctors and patients alike: what are the chances of an outbreak for a specific disease? Can individual risks be managed and if so how? Which drugs are available and how should they be applied? Could a new drug be tailored to an individual’s genetic predisposition fast and in an affordable way? In order to provide answers and real-life value to patients, the rapid evolvement of novel computing approaches for analyzing big data in systems genomics has to be accompanied by an equally strong effort to develop next-generation DNA-sequencing and next-generation drug screening and design platforms. In that context lab-on-a-chip devices utilizing nanopore- and nanochannel based resistive pulse-sensing technology for DNA-sequencing and protein screening applications occupy a key role. This paper describes the status quo of resistive pulse sensing technology for these two application areas with a special focus on current technology trends and challenges ahead.

Label-free screening of single biomolecules through resistive pulse sensing technology for precision medicine applications.

PubMed

Harrer, S; Kim, S C; Schieber, C; Kannam, S; Gunn, N; Moore, S; Scott, D; Bathgate, R; Skafidas, S; Wagner, J M

2015-05-08

Employing integrated nano- and microfluidic circuits for detecting and characterizing biological compounds through resistive pulse sensing technology is a vibrant area of research at the interface of biotechnology and nanotechnology. Resistive pulse sensing platforms can be customized to study virtually any particle of choice which can be threaded through a fluidic channel and enable label-free single-particle interrogation with the primary read-out signal being an electric current fingerprint. The ability to perform label-free molecular screening with single-molecule and even single binding site resolution makes resistive pulse sensing technology a powerful tool for analyzing the smallest units of biological systems and how they interact with each other on a molecular level. This task is at the core of experimental systems biology and in particular 'omics research which in combination with next-generation DNA-sequencing and next-generation drug discovery and design forms the foundation of a novel disruptive medical paradigm commonly referred to as personalized medicine or precision medicine. DNA-sequencing has approached the 1000-Dollar-Genome milestone allowing for decoding a complete human genome with unmatched speed and at low cost. Increased sequencing efficiency yields massive amounts of genomic data. Analyzing this data in combination with medical and biometric health data eventually enables understanding the pathways from individual genes to physiological functions. Access to this information triggers fundamental questions for doctors and patients alike: what are the chances of an outbreak for a specific disease? Can individual risks be managed and if so how? Which drugs are available and how should they be applied? Could a new drug be tailored to an individual's genetic predisposition fast and in an affordable way? In order to provide answers and real-life value to patients, the rapid evolvement of novel computing approaches for analyzing big data in systems genomics has to be accompanied by an equally strong effort to develop next-generation DNA-sequencing and next-generation drug screening and design platforms. In that context lab-on-a-chip devices utilizing nanopore- and nanochannel based resistive pulse-sensing technology for DNA-sequencing and protein screening applications occupy a key role. This paper describes the status quo of resistive pulse sensing technology for these two application areas with a special focus on current technology trends and challenges ahead.

Estimating genotype error rates from high-coverage next-generation sequence data.

PubMed

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.

Rapid Bacterial Whole-Genome Sequencing to Enhance Diagnostic and Public Health Microbiology

PubMed Central

Reuter, Sandra; Ellington, Matthew J.; Cartwright, Edward J. P.; Köser, Claudio U.; Török, M. Estée; Gouliouris, Theodore; Harris, Simon R.; Brown, Nicholas M.; Holden, Matthew T. G.; Quail, Mike; Parkhill, Julian; Smith, Geoffrey P.; Bentley, Stephen D.; Peacock, Sharon J.

2014-01-01

IMPORTANCE The latest generation of benchtop DNA sequencing platforms can provide an accurate whole-genome sequence (WGS) for a broad range of bacteria in less than a day. These could be used to more effectively contain the spread of multidrug-resistant pathogens. OBJECTIVE To compare WGS with standard clinical microbiology practice for the investigation of nosocomial outbreaks caused by multidrug-resistant bacteria, the identification of genetic determinants of antimicrobial resistance, and typing of other clinically important pathogens. DESIGN, SETTING, AND PARTICIPANTS A laboratory-based study of hospital inpatients with a range of bacterial infections at Cambridge University Hospitals NHS Foundation Trust, a secondary and tertiary referral center in England, comparing WGS with standard diagnostic microbiology using stored bacterial isolates and clinical information. MAIN OUTCOMES AND MEASURES Specimens were taken and processed as part of routine clinical care, and cultured isolates stored and referred for additional reference laboratory testing as necessary. Isolates underwent DNA extraction and library preparation prior to sequencing on the Illumina MiSeq platform. Bioinformatic analyses were performed by persons blinded to the clinical, epidemiologic, and antimicrobial susceptibility data. RESULTS We investigated 2 putative nosocomial outbreaks, one caused by vancomycin-resistant Enterococcus faecium and the other by carbapenem-resistant Enterobacter cloacae; WGS accurately discriminated between outbreak and nonoutbreak isolates and was superior to conventional typing methods. We compared WGS with standard methods for the identification of the mechanism of carbapenem resistance in a range of gram-negative bacteria (Acinetobacter baumannii, E cloacae, Escherichia coli, and Klebsiella pneumoniae). This demonstrated concordance between phenotypic and genotypic results, and the ability to determine whether resistance was attributable to the presence of carbapenemases or other resistance mechanisms. Whole-genome sequencing was used to recapitulate reference laboratory typing of clinical isolates of Neisseria meningitidis and to provide extended phylogenetic analyses of these. CONCLUSIONS AND RELEVANCE The speed, accuracy, and depth of information provided by WGS platforms to confirm or refute outbreaks in hospitals and the community, and to accurately define transmission of multidrug-resistant and other organisms, represents an important advance. PMID:23857503

Microbial forensics: fiber optic microarray subtyping of Bacillus anthracis

NASA Astrophysics Data System (ADS)

Shepard, Jason R. E.

2009-05-01

The past decade has seen increased development and subsequent adoption of rapid molecular techniques involving DNA analysis for detection of pathogenic microorganisms, also termed microbial forensics. The continued accumulation of microbial sequence information in genomic databases now better positions the field of high-throughput DNA analysis to proceed in a more manageable fashion. The potential to build off of these databases exists as technology continues to develop, which will enable more rapid, cost effective analyses. This wealth of genetic information, along with new technologies, has the potential to better address some of the current problems and solve the key issues involved in DNA analysis of pathogenic microorganisms. To this end, a high density fiber optic microarray has been employed, housing numerous DNA sequences simultaneously for detection of various pathogenic microorganisms, including Bacillus anthracis, among others. Each organism is analyzed with multiple sequences and can be sub-typed against other closely related organisms. For public health labs, real-time PCR methods have been developed as an initial preliminary screen, but culture and growth are still considered the gold standard. Technologies employing higher throughput than these standard methods are better suited to capitalize on the limitless potential garnered from the sequence information. Microarray analyses are one such format positioned to exploit this potential, and our array platform is reusable, allowing repetitive tests on a single array, providing an increase in throughput and decrease in cost, along with a certainty of detection, down to the individual strain level.

Comparing sequencing assays and human-machine analyses in actionable genomics for glioblastoma

PubMed Central

Wrzeszczynski, Kazimierz O.; Frank, Mayu O.; Koyama, Takahiko; Rhrissorrakrai, Kahn; Robine, Nicolas; Utro, Filippo; Emde, Anne-Katrin; Chen, Bo-Juen; Arora, Kanika; Shah, Minita; Vacic, Vladimir; Norel, Raquel; Bilal, Erhan; Bergmann, Ewa A.; Moore Vogel, Julia L.; Bruce, Jeffrey N.; Lassman, Andrew B.; Canoll, Peter; Grommes, Christian; Harvey, Steve; Parida, Laxmi; Michelini, Vanessa V.; Zody, Michael C.; Jobanputra, Vaidehi; Royyuru, Ajay K.

2017-01-01

Objective: To analyze a glioblastoma tumor specimen with 3 different platforms and compare potentially actionable calls from each. Methods: Tumor DNA was analyzed by a commercial targeted panel. In addition, tumor-normal DNA was analyzed by whole-genome sequencing (WGS) and tumor RNA was analyzed by RNA sequencing (RNA-seq). The WGS and RNA-seq data were analyzed by a team of bioinformaticians and cancer oncologists, and separately by IBM Watson Genomic Analytics (WGA), an automated system for prioritizing somatic variants and identifying drugs. Results: More variants were identified by WGS/RNA analysis than by targeted panels. WGA completed a comparable analysis in a fraction of the time required by the human analysts. Conclusions: The development of an effective human-machine interface in the analysis of deep cancer genomic datasets may provide potentially clinically actionable calls for individual patients in a more timely and efficient manner than currently possible. ClinicalTrials.gov identifier: NCT02725684. PMID:28740869

Droplet-based pyrosequencing using digital microfluidics.

PubMed

Boles, Deborah J; Benton, Jonathan L; Siew, Germaine J; Levy, Miriam H; Thwar, Prasanna K; Sandahl, Melissa A; Rouse, Jeremy L; Perkins, Lisa C; Sudarsan, Arjun P; Jalili, Roxana; Pamula, Vamsee K; Srinivasan, Vijay; Fair, Richard B; Griffin, Peter B; Eckhardt, Allen E; Pollack, Michael G

2011-11-15

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., "sample-to-sequence" capability) could eventually be achieved using this low-cost platform.

MytiBase: a knowledgebase of mussel (M. galloprovincialis) transcribed sequences

PubMed Central

Venier, Paola; De Pittà, Cristiano; Bernante, Filippo; Varotto, Laura; De Nardi, Barbara; Bovo, Giuseppe; Roch, Philippe; Novoa, Beatriz; Figueras, Antonio; Pallavicini, Alberto; Lanfranchi, Gerolamo

2009-01-01

Background Although Bivalves are among the most studied marine organisms due to their ecological role, economic importance and use in pollution biomonitoring, very little information is available on the genome sequences of mussels. This study reports the functional analysis of a large-scale Expressed Sequence Tag (EST) sequencing from different tissues of Mytilus galloprovincialis (the Mediterranean mussel) challenged with toxic pollutants, temperature and potentially pathogenic bacteria. Results We have constructed and sequenced seventeen cDNA libraries from different Mediterranean mussel tissues: gills, digestive gland, foot, anterior and posterior adductor muscle, mantle and haemocytes. A total of 24,939 clones were sequenced from these libraries generating 18,788 high-quality ESTs which were assembled into 2,446 overlapping clusters and 4,666 singletons resulting in a total of 7,112 non-redundant sequences. In particular, a high-quality normalized cDNA library (Nor01) was constructed as determined by the high rate of gene discovery (65.6%). Bioinformatic screening of the non-redundant M. galloprovincialis sequences identified 159 microsatellite-containing ESTs. Clusters, consensuses, related similarities and gene ontology searches have been organized in a dedicated, searchable database . Conclusion We defined the first species-specific catalogue of M. galloprovincialis ESTs including 7,112 unique transcribed sequences. Putative microsatellite markers were identified. This annotated catalogue represents a valuable platform for expression studies, marker validation and genetic linkage analysis for investigations in the biology of Mediterranean mussels. PMID:19203376

Launching genomics into the cloud: deployment of Mercury, a next generation sequence analysis pipeline.

PubMed

Reid, Jeffrey G; Carroll, Andrew; Veeraraghavan, Narayanan; Dahdouli, Mahmoud; Sundquist, Andreas; English, Adam; Bainbridge, Matthew; White, Simon; Salerno, William; Buhay, Christian; Yu, Fuli; Muzny, Donna; Daly, Richard; Duyk, Geoff; Gibbs, Richard A; Boerwinkle, Eric

2014-01-29

Massively parallel DNA sequencing generates staggering amounts of data. Decreasing cost, increasing throughput, and improved annotation have expanded the diversity of genomics applications in research and clinical practice. This expanding scale creates analytical challenges: accommodating peak compute demand, coordinating secure access for multiple analysts, and sharing validated tools and results. To address these challenges, we have developed the Mercury analysis pipeline and deployed it in local hardware and the Amazon Web Services cloud via the DNAnexus platform. Mercury is an automated, flexible, and extensible analysis workflow that provides accurate and reproducible genomic results at scales ranging from individuals to large cohorts. By taking advantage of cloud computing and with Mercury implemented on the DNAnexus platform, we have demonstrated a powerful combination of a robust and fully validated software pipeline and a scalable computational resource that, to date, we have applied to more than 10,000 whole genome and whole exome samples.

Ultrasensitive sensing platform for platelet-derived growth factor BB detection based on layered molybdenum selenide-graphene composites and Exonuclease III assisted signal amplification.

PubMed

Huang, Ke-Jing; Shuai, Hong-Lei; Zhang, Ji-Zong

2016-03-15

A highly sensitive and ultrasensitive electrochemical aptasensor for platelet-derived growth factor BB (PDGF-BB) detection is fabricated based on layered molybdenum selenide-graphene (MoSe2-Gr) composites and Exonuclease III (Exo III)-aided signal amplification. MoSe2-Gr is prepared by a simple hydrothermal method and used as a promising sensing platform. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity is limited on the duplex DNAs with more than 4 mismatched terminal bases at 3' ends. Herein, aptamer and complementary DNA (cDNA) sequences are designed with four thymine bases on 3' ends. In the presence of target protein, the aptamer associates with it and facilitates the formation of duplex DNA between cDNA and signal DNA. The duplex DNA then is digested by Exo III and releases cDNA, which hybridizes with signal DNA to perform a new cleavage process. Nevertheless, in the absence of target protein, the aptamer hybridizes with cDNA will inhibit the Exo III-assisted nucleotides cleavage. The signal DNA then hybridizes with capture DNA on the electrode. Subsequently, horse radish peroxidase is fixed on electrode by avidin-biotin reaction and then catalyzes hydrogen peroxide and hydroquinone to produce electrochemical response. Therefore, a bridge can be established between the concentration of target protein and the degree of the attenuation of the obtained signal, providing a quantitative measure of target protein with a broad detection range of 0.0001-1 nM and a detection limit of 20 fM. Copyright © 2015 Elsevier B.V. All rights reserved.

Next-generation sequencing: hype and hope for development of personalized radiation therapy?

PubMed

Tinhofer, Ingeborg; Niehr, Franziska; Konschak, Robert; Liebs, Sandra; Munz, Matthias; Stenzinger, Albrecht; Weichert, Wilko; Keilholz, Ulrich; Budach, Volker

2015-08-28

The introduction of next-generation sequencing (NGS) in the field of cancer research has boosted worldwide efforts of genome-wide personalized oncology aiming at identifying predictive biomarkers and novel actionable targets. Despite considerable progress in understanding the molecular biology of distinct cancer entities by the use of this revolutionary technology and despite contemporaneous innovations in drug development, translation of NGS findings into improved concepts for cancer treatment remains a challenge. The aim of this article is to describe shortly the NGS platforms for DNA sequencing and in more detail key achievements and unresolved hurdles. A special focus will be given on potential clinical applications of this innovative technique in the field of radiation oncology.

Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection.

PubMed

Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

2016-07-29

Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world's attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg(2+) ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species.

Integrating a DNA Strand Displacement Reaction with a Whispering Gallery Mode Sensor for Label-Free Mercury (II) Ion Detection

PubMed Central

Wu, Fengchi; Wu, Yuqiang; Niu, Zhongwei; Vollmer, Frank

2016-01-01

Mercury is an extremely toxic chemical pollutant of our environment. It has attracted the world’s attention due to its high mobility and the ease with which it accumulates in organisms. Sensitive devices and methods specific for detecting mercury ions are, hence, in great need. Here, we have integrated a DNA strand displacement reaction with a whispering gallery mode (WGM) sensor for demonstrating the detection of Hg2+ ions. Our approach relies on the displacement of a DNA hairpin structure, which forms after the binding of mercury ions to an aptamer DNA sequence. The strand displacement reaction of the DNA aptamer provides highly specific and quantitative means for determining the mercury ion concentration on a label-free WGM sensor platform. Our approach also shows the possibility for manipulating the kinetics of a strand displacement reaction with specific ionic species. PMID:27483277

[Detection of pathogenic mutations in Marfan syndrome by targeted next-generation semiconductor sequencing].

PubMed

Lu, Chaoxia; Wu, Wei; Xiao, Jifang; Meng, Yan; Zhang, Shuyang; Zhang, Xue

2013-06-01

To detect pathogenic mutations in Marfan syndrome (MFS) using an Ion Torrent Personal Genome Machine (PGM) and to validate the result of targeted next-generation semiconductor sequencing for the diagnosis of genetic disorders. Peripheral blood samples were collected from three MFS patients and a normal control with informed consent. Genomic DNA was isolated by standard method and then subjected to targeted sequencing using an Ion Ampliseq(TM) Inherited Disease Panel. Three multiplex PCR reactions were carried out to amplify the coding exons of 328 genes including FBN1, TGFBR1 and TGFBR2. DNA fragments from different samples were ligated with barcoded sequencing adaptors. Template preparation and emulsion PCR, and Ion Sphere Particles enrichment were carried out using an Ion One Touch system. The ion sphere particles were sequenced on a 318 chip using the PGM platform. Data from the PGM runs were processed using an Ion Torrent Suite 3.2 software to generate sequence reads. After sequence alignment and extraction of SNPs and indels, all the variants were filtered against dbSNP137. DNA sequences were visualized with an Integrated Genomics Viewer. The most likely disease-causing variants were analyzed by Sanger sequencing. The PGM sequencing has yielded an output of 855.80 Mb, with a > 100 × median sequencing depth and a coverage of > 98% for the targeted regions in all the four samples. After data analysis and database filtering, one known missense mutation (p.E1811K) and two novel premature termination mutations (p.E2264X and p.L871FfsX23) in the FBN1 gene were identified in the three MFS patients. All mutations were verified by conventional Sanger sequencing. Pathogenic FBN1 mutations have been identified in all patients with MFS, indicating that the targeted next-generation sequencing on the PGM sequencers can be applied for accurate and high-throughput testing of genetic disorders.

An optimized method for high quality DNA extraction from microalga Prototheca wickerhamii for genome sequencing.

PubMed

Jagielski, Tomasz; Gawor, Jan; Bakuła, Zofia; Zuchniewicz, Karolina; Żak, Iwona; Gromadka, Robert

2017-01-01

The complex cell wall structure of algae often precludes efficient extraction of their genetic material. The purpose of this study was to design a next-generation sequencing-suitable DNA isolation method for unicellular, achlorophyllous, yeast-like microalgae of the genus Prototheca , the only known plant pathogens of both humans and animals. The effectiveness of the newly proposed scheme was compared with five other, previously described methods, commonly used for DNA isolation from plants and/or yeasts, available either as laboratory-developed, in-house assays, based on liquid nitrogen grinding or different enzymatic digestion, or as commercially manufactured kits. All five, previously described, isolation assays yielded DNA concentrations lower than those obtained with the new method, averaging 16.15 ± 25.39 vs 74.2 ± 0.56 ng/µL, respectively. The new method was also superior in terms of DNA purity, as measured by A260/A280 (-0.41 ± 4.26 vs 2.02 ± 0.03), and A260/A230 (1.20 ± 1.12 vs 1.97 ± 0.07) ratios. Only the liquid nitrogen-based method yielded DNA of comparable quantity (60.96 ± 0.16 ng/µL) and quality (A260/A280 = 2.08 ± 0.02; A260/A230 = 2.23 ± 0.26). Still, the new method showed higher integrity, which was best illustrated upon electrophoretic analysis. Genomic DNA of Prototheca wickerhamii POL-1 strain isolated with the protocol herein proposed was successfully sequenced on the Illumina MiSeq platform. A new method for DNA isolation from Prototheca algae is described. The method, whose protocol involves glass beads pulverization and cesium chloride (CsCl) density gradient centrifugation, was demonstrated superior over the other common assays in terms of DNA quantity and quality. The method is also the first to offer the possibility of preparation of DNA template suitable for whole genome sequencing of Prototheca spp.

A genosensor for detection of consensus DNA sequence of Dengue virus using ZnO/Pt-Pd nanocomposites.

PubMed

Singhal, Chaitali; Pundir, C S; Narang, Jagriti

2017-11-15

An electrochemical genosensor based on Zinc oxide/platinum-palladium (ZnO/Pt-Pd) modified fluorine doped tin oxide (FTO) glass plate was fabricated for detection of consensus DNA sequence of Dengue virus (DENV) using methylene blue (MB) as an intercalating agent. To achieve it, probe DNA (PDNA) was immobilized on the surface of ZnO/Pt-Pd nanocomposites modified FTO electrode. The synthesized nano-composites were characterized by high resolution transmission electron microscopy (HRTEM), energy dispersive X-ray analysis (EDX), atomic force microscopy (AFM), scanning electron microscopy (SEM), UV-Vis spectroscopy, X-ray diffraction (XRD) analysis and Fourier transform infra-red (FTIR) spectroscopy. This PDNA modified electrode (PDNA/ZnO/Pt-Pd/FTO) served as a signal amplification platform for the detection of the target hybridized DNA (TDNA). The hybridization between PDNA and TDNA was detected by reduction in current, generated by interaction of anionic mediator, i.e., methylene blue (MB) with free guanine (3'G) of ssDNA. The sensor showed a dynamic linear range of 1 × 10 -6 M to 100 × 10 -6 M with LOD as 4.3 × 10 -5 M and LOQ as 9.5 × 10 -5 M. Till date, majorly serotype specific biosensors for dengue detection have been developed. The genosensor reported here eliminates the possibility of false result as in case of serotype specific DNA sensor. This is the report where conserved sequences present in all the serotypes of Dengue virus has been employed for fabrication of a genosensor. Copyright © 2017 Elsevier B.V. All rights reserved.

Genomes by design

PubMed Central

Haimovich, Adrian D.; Muir, Paul; Isaacs, Farren J.

2016-01-01

Next-generation DNA sequencing has revealed the complete genome sequences of numerous organisms, establishing a fundamental and growing understanding of genetic variation and phenotypic diversity. Engineering at the gene, network and whole-genome scale aims to introduce targeted genetic changes both to explore emergent phenotypes and to introduce new functionalities. Expansion of these approaches into massively parallel platforms establishes the ability to generate targeted genome modifications, elucidating causal links between genotype and phenotype, as well as the ability to design and reprogramme organisms. In this Review, we explore techniques and applications in genome engineering, outlining key advances and defining challenges. PMID:26260262

Noninvasive prenatal diagnosis of common aneuploidies by semiconductor sequencing

PubMed Central

Liao, Can; Yin, Ai-hua; Peng, Chun-fang; Fu, Fang; Yang, Jie-xia; Li, Ru; Chen, Yang-yi; Luo, Dong-hong; Zhang, Yong-ling; Ou, Yan-mei; Li, Jian; Wu, Jing; Mai, Ming-qin; Hou, Rui; Wu, Frances; Luo, Hongrong; Li, Dong-zhi; Liu, Hai-liang; Zhang, Xiao-zhuang; Zhang, Kang

2014-01-01

Massively parallel sequencing (MPS) of cell-free fetal DNA from maternal plasma has revolutionized our ability to perform noninvasive prenatal diagnosis. This approach avoids the risk of fetal loss associated with more invasive diagnostic procedures. The present study developed an effective method for noninvasive prenatal diagnosis of common chromosomal aneuploidies using a benchtop semiconductor sequencing platform (SSP), which relies on the MPS platform but offers advantages over existing noninvasive screening techniques. A total of 2,275 pregnant subjects was included in the study; of these, 515 subjects who had full karyotyping results were used in a retrospective analysis, and 1,760 subjects without karyotyping were analyzed in a prospective study. In the retrospective study, all 55 fetal trisomy 21 cases were identified using the SSP with a sensitivity and specificity of 99.94% and 99.46%, respectively. The SSP also detected 16 trisomy 18 cases with 100% sensitivity and 99.24% specificity and 3 trisomy 13 cases with 100% sensitivity and 100% specificity. Furthermore, 15 fetuses with sex chromosome aneuploidies (10 45,X, 2 47,XYY, 2 47,XXX, and 1 47,XXY) were detected. In the prospective study, nine fetuses with trisomy 21, three with trisomy 18, three with trisomy 13, and one with 45,X were detected. To our knowledge, this is the first large-scale clinical study to systematically identify chromosomal aneuploidies based on cell-free fetal DNA using the SSP and provides an effective strategy for large-scale noninvasive screening for chromosomal aneuploidies in a clinical setting. PMID:24799683

Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing.

PubMed

He, Bifang; Tjhung, Katrina F; Bennett, Nicholas J; Chou, Ying; Rau, Andrea; Huang, Jian; Derda, Ratmir

2018-01-19

Understanding the composition of a genetically-encoded (GE) library is instrumental to the success of ligand discovery. In this manuscript, we investigate the bias in GE-libraries of linear, macrocyclic and chemically post-translationally modified (cPTM) tetrapeptides displayed on the M13KE platform, which are produced via trinucleotide cassette synthesis (19 codons) and NNK-randomized codon. Differential enrichment of synthetic DNA {S}, ligated vector {L} (extension and ligation of synthetic DNA into the vector), naïve libraries {N} (transformation of the ligated vector into the bacteria followed by expression of the library for 4.5 hours to yield a "naïve" library), and libraries chemically modified by aldehyde ligation and cysteine macrocyclization {M} characterized by paired-end deep sequencing, detected a significant drop in diversity in {L} → {N}, but only a minor compositional difference in {S} → {L} and {N} → {M}. Libraries expressed at the N-terminus of phage protein pIII censored positively charged amino acids Arg and Lys; libraries expressed between pIII domains N1 and N2 overcame Arg/Lys-censorship but introduced new bias towards Gly and Ser. Interrogation of biases arising from cPTM by aldehyde ligation and cysteine macrocyclization unveiled censorship of sequences with Ser/Phe. Analogous analysis can be used to explore library diversity in new display platforms and optimize cPTM of these libraries.

Electrocatalysis in DNA Sensors

PubMed Central

Furst, Ariel; Hill, Michael G.; Barton, Jacqueline K.

2014-01-01

Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology. PMID:25435647

Electrocatalysis in DNA Sensors.

PubMed

Furst, Ariel; Hill, Michael G; Barton, Jacqueline K

2014-12-14

Electrocatalysis is often thought of solely in the inorganic realm, most often applied to energy conversion in fuel cells. However, the ever-growing field of bioelectrocatalysis has made great strides in advancing technology for both biofuel cells as well as biological detection platforms. Within the context of bioelectrocatalytic detection systems, DNA-based platforms are especially prevalent. One subset of these platforms, the one we have developed, takes advantage of the inherent charge transport properties of DNA. Electrocatalysis coupled with DNA-mediated charge transport has enabled specific and sensitive detection of lesions, mismatches and DNA-binding proteins. Even greater signal amplification from these platforms is now being achieved through the incorporation of a secondary electrode to the platform both for patterning DNA arrays and for detection. Here, we describe the evolution of this new DNA sensor technology.

Deep Sequencing of T-cell Receptor DNA as a Biomarker of Clonally Expanded TILs in Breast Cancer after Immunotherapy.

PubMed

Page, David B; Yuan, Jianda; Redmond, David; Wen, Y Hanna; Durack, Jeremy C; Emerson, Ryan; Solomon, Stephen; Dong, Zhiwan; Wong, Phillip; Comstock, Christopher; Diab, Adi; Sung, Janice; Maybody, Majid; Morris, Elizabeth; Brogi, Edi; Morrow, Monica; Sacchini, Virgilio; Elemento, Olivier; Robins, Harlan; Patil, Sujata; Allison, James P; Wolchok, Jedd D; Hudis, Clifford; Norton, Larry; McArthur, Heather L

2016-10-01

In early-stage breast cancer, the degree of tumor-infiltrating lymphocytes (TIL) predicts response to chemotherapy and overall survival. Combination immunotherapy with immune checkpoint antibody plus tumor cryoablation can induce lymphocytic infiltrates and improve survival in mice. We used T-cell receptor (TCR) DNA sequencing to evaluate both the effect of cryoimmunotherapy in humans and the feasibility of TCR sequencing in early-stage breast cancer. In a pilot clinical trial, 18 women with early-stage breast cancer were treated preoperatively with cryoablation, single-dose anti-CTLA-4 (ipilimumab), or cryoablation + ipilimumab. TCRs within serially collected peripheral blood and tumor tissue were sequenced. In baseline tumor tissues, T-cell density as measured by TCR sequencing correlated with TIL scores obtained by hematoxylin and eosin (H&E) staining. However, tumors with little or no lymphocytes by H&E contained up to 3.6 × 10 6 TCR DNA sequences, highlighting the sensitivity of the ImmunoSEQ platform. In this dataset, ipilimumab increased intratumoral T-cell density over time, whereas cryoablation ± ipilimumab diversified and remodeled the intratumoral T-cell clonal repertoire. Compared with monotherapy, cryoablation plus ipilimumab was associated with numerically greater numbers of peripheral blood and intratumoral T-cell clones expanding robustly following therapy. In conclusion, TCR sequencing correlates with H&E lymphocyte scoring and provides additional information on clonal diversity. These findings support further study of the use of TCR sequencing as a biomarker for T-cell responses to therapy and for the study of cryoimmunotherapy in early-stage breast cancer. Cancer Immunol Res; 4(10); 835-44. ©2016 AACR. ©2016 American Association for Cancer Research.

Bias-Corrected Targeted Next-Generation Sequencing for Rapid, Multiplexed Detection of Actionable Alterations in Cell-Free DNA from Advanced Lung Cancer Patients.

PubMed

Paweletz, Cloud P; Sacher, Adrian G; Raymond, Chris K; Alden, Ryan S; O'Connell, Allison; Mach, Stacy L; Kuang, Yanan; Gandhi, Leena; Kirschmeier, Paul; English, Jessie M; Lim, Lee P; Jänne, Pasi A; Oxnard, Geoffrey R

2016-02-15

Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. NGS could identify mutations present in DNA dilutions at ≥ 0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. ©2015 American Association for Cancer Research.

Bias-corrected targeted next-generation sequencing for rapid, multiplexed detection of actionable alterations in cell-free DNA from advanced lung cancer patients

PubMed Central

Paweletz, Cloud P.; Sacher, Adrian G.; Raymond, Chris K.; Alden, Ryan S.; O'Connell, Allison; Mach, Stacy L.; Kuang, Yanan; Gandhi, Leena; Kirschmeier, Paul; English, Jessie M.; Lim, Lee P.; Jänne, Pasi A.; Oxnard, Geoffrey R.

2015-01-01

Purpose Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care, however comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). Experimental Design An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. Results NGS could identify mutations present in DNA dilutions at ≥0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Conclusion Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. PMID:26459174

Bicycle: a bioinformatics pipeline to analyze bisulfite sequencing data.

PubMed

Graña, Osvaldo; López-Fernández, Hugo; Fdez-Riverola, Florentino; González Pisano, David; Glez-Peña, Daniel

2018-04-15

High-throughput sequencing of bisulfite-converted DNA is a technique used to measure DNA methylation levels. Although a considerable number of computational pipelines have been developed to analyze such data, none of them tackles all the peculiarities of the analysis together, revealing limitations that can force the user to manually perform additional steps needed for a complete processing of the data. This article presents bicycle, an integrated, flexible analysis pipeline for bisulfite sequencing data. Bicycle analyzes whole genome bisulfite sequencing data, targeted bisulfite sequencing data and hydroxymethylation data. To show how bicycle overtakes other available pipelines, we compared them on a defined number of features that are summarized in a table. We also tested bicycle with both simulated and real datasets, to show its level of performance, and compared it to different state-of-the-art methylation analysis pipelines. Bicycle is publicly available under GNU LGPL v3.0 license at http://www.sing-group.org/bicycle. Users can also download a customized Ubuntu LiveCD including bicycle and other bisulfite sequencing data pipelines compared here. In addition, a docker image with bicycle and its dependencies, which allows a straightforward use of bicycle in any platform (e.g. Linux, OS X or Windows), is also available. ograna@cnio.es or dgpena@uvigo.es. Supplementary data are available at Bioinformatics online.

IMG/VR: a database of cultured and uncultured DNA Viruses and retroviruses.

PubMed

Paez-Espino, David; Chen, I-Min A; Palaniappan, Krishna; Ratner, Anna; Chu, Ken; Szeto, Ernest; Pillay, Manoj; Huang, Jinghua; Markowitz, Victor M; Nielsen, Torben; Huntemann, Marcel; K Reddy, T B; Pavlopoulos, Georgios A; Sullivan, Matthew B; Campbell, Barbara J; Chen, Feng; McMahon, Katherine; Hallam, Steve J; Denef, Vincent; Cavicchioli, Ricardo; Caffrey, Sean M; Streit, Wolfgang R; Webster, John; Handley, Kim M; Salekdeh, Ghasem H; Tsesmetzis, Nicolas; Setubal, Joao C; Pope, Phillip B; Liu, Wen-Tso; Rivers, Adam R; Ivanova, Natalia N; Kyrpides, Nikos C

2017-01-04

Viruses represent the most abundant life forms on the planet. Recent experimental and computational improvements have led to a dramatic increase in the number of viral genome sequences identified primarily from metagenomic samples. As a result of the expanding catalog of metagenomic viral sequences, there exists a need for a comprehensive computational platform integrating all these sequences with associated metadata and analytical tools. Here we present IMG/VR (https://img.jgi.doe.gov/vr/), the largest publicly available database of 3908 isolate reference DNA viruses with 264 413 computationally identified viral contigs from >6000 ecologically diverse metagenomic samples. Approximately half of the viral contigs are grouped into genetically distinct quasi-species clusters. Microbial hosts are predicted for 20 000 viral sequences, revealing nine microbial phyla previously unreported to be infected by viruses. Viral sequences can be queried using a variety of associated metadata, including habitat type and geographic location of the samples, or taxonomic classification according to hallmark viral genes. IMG/VR has a user-friendly interface that allows users to interrogate all integrated data and interact by comparing with external sequences, thus serving as an essential resource in the viral genomics community. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Accuracy of the high-throughput amplicon sequencing to identify species within the genus Aspergillus.

PubMed

Lee, Seungeun; Yamamoto, Naomichi

2015-12-01

This study characterized the accuracy of high-throughput amplicon sequencing to identify species within the genus Aspergillus. To this end, we sequenced the internal transcribed spacer 1 (ITS1), β-tubulin (BenA), and calmodulin (CaM) gene encoding sequences as DNA markers from eight reference Aspergillus strains with known identities using 300-bp sequencing on the Illumina MiSeq platform, and compared them with the BLASTn outputs. The identifications with the sequences longer than 250 bp were accurate at the section rank, with some ambiguities observed at the species rank due to mostly cross detection of sibling species. Additionally, in silico analysis was performed to predict the identification accuracy for all species in the genus Aspergillus, where 107, 210, and 187 species were predicted to be identifiable down to the species rank based on ITS1, BenA, and CaM, respectively. Finally, air filter samples were analysed to quantify the relative abundances of Aspergillus species in outdoor air. The results were reproducible across biological duplicates both at the species and section ranks, but not strongly correlated between ITS1 and BenA, suggesting the Aspergillus detection can be taxonomically biased depending on the selection of the DNA markers and/or primers. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Mitochondrial and chloroplast phylogeography of Picea crassifolia Kom. (Pinaceae) in the Qinghai-Tibetan Plateau and adjacent highlands.

PubMed

Meng, Lihua; Yang, Rui; Abbott, Richard J; Miehe, Georg; Hu, Tianhua; Liu, Jianquan

2007-10-01

The disjunct distribution of forests in the Qinghai-Tibetan Plateau (QTP) and adjacent Helan Shan and Daqing Shan highlands provides an excellent model to examine vegetation shifts, glacial refugia and gene flow of key species in this complex landscape region in response to past climatic oscillations and human disturbance. In this study, we examined maternally inherited mitochondrial DNA (nad1 intron b/c and nad5 intron 1) and paternally inherited chloroplast DNA (trnC-trnD) sequence variation within a dominant forest species, Picea crassifolia Kom. We recovered nine mitotypes and two chlorotypes in a survey of 442 individuals from 32 populations sampled throughout the species' range. Significant mitochondrial DNA population subdivision was detected (G(ST) = 0.512; N(ST) = 0.679), suggesting low levels of recurrent gene flow through seeds among populations and significant phylogeographical structure (N(ST) > GST, P < 0.05). Plateau haplotypes differed in sequence from those in the adjacent highlands, suggesting a long period of allopatric fragmentation between the species in the two regions and the presence of independent refugia in each region during Quaternary glaciations. On the QTP platform, all but one of the disjunct populations surveyed were fixed for the same mitotype, while most populations at the plateau edge contained more than one haplotype with the mitotype that was fixed in plateau platform populations always present at high frequency. This distribution pattern suggests that present-day disjunct populations on the QTP platform experienced a common recolonization history. The same phylogeographical pattern, however, was not detected for paternally inherited chloroplast DNA haplotypes. Two chlorotypes were distributed throughout the range of the species with little geographical population differentiation (G(ST) = N(ST) = 0.093). This provides evidence for highly efficient pollen-mediated gene flow among isolated forest patches, both within and between the QTP and adjacent highland populations. A lack of isolation to pollen-mediated gene flow between forests on the QTP and adjacent highlands is surprising given that the Tengger Desert has been a geographical barrier between these two regions for approximately the last 1.8 million years.

Environmental microbiology through the lens of high-throughput DNA sequencing: synopsis of current platforms and bioinformatics approaches.

PubMed

Logares, Ramiro; Haverkamp, Thomas H A; Kumar, Surendra; Lanzén, Anders; Nederbragt, Alexander J; Quince, Christopher; Kauserud, Håvard

2012-10-01

The incursion of High-Throughput Sequencing (HTS) in environmental microbiology brings unique opportunities and challenges. HTS now allows a high-resolution exploration of the vast taxonomic and metabolic diversity present in the microbial world, which can provide an exceptional insight on global ecosystem functioning, ecological processes and evolution. This exploration has also economic potential, as we will have access to the evolutionary innovation present in microbial metabolisms, which could be used for biotechnological development. HTS is also challenging the research community, and the current bottleneck is present in the data analysis side. At the moment, researchers are in a sequence data deluge, with sequencing throughput advancing faster than the computer power needed for data analysis. However, new tools and approaches are being developed constantly and the whole process could be depicted as a fast co-evolution between sequencing technology, informatics and microbiologists. In this work, we examine the most popular and recently commercialized HTS platforms as well as bioinformatics methods for data handling and analysis used in microbial metagenomics. This non-exhaustive review is intended to serve as a broad state-of-the-art guide to researchers expanding into this rapidly evolving field. Copyright © 2012 Elsevier B.V. All rights reserved.

Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi

2016-03-01

Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

ampliMethProfiler: a pipeline for the analysis of CpG methylation profiles of targeted deep bisulfite sequenced amplicons.

PubMed

Scala, Giovanni; Affinito, Ornella; Palumbo, Domenico; Florio, Ermanno; Monticelli, Antonella; Miele, Gennaro; Chiariotti, Lorenzo; Cocozza, Sergio

2016-11-25

CpG sites in an individual molecule may exist in a binary state (methylated or unmethylated) and each individual DNA molecule, containing a certain number of CpGs, is a combination of these states defining an epihaplotype. Classic quantification based approaches to study DNA methylation are intrinsically unable to fully represent the complexity of the underlying methylation substrate. Epihaplotype based approaches, on the other hand, allow methylation profiles of cell populations to be studied at the single molecule level. For such investigations, next-generation sequencing techniques can be used, both for quantitative and for epihaplotype analysis. Currently available tools for methylation analysis lack output formats that explicitly report CpG methylation profiles at the single molecule level and that have suited statistical tools for their interpretation. Here we present ampliMethProfiler, a python-based pipeline for the extraction and statistical epihaplotype analysis of amplicons from targeted deep bisulfite sequencing of multiple DNA regions. ampliMethProfiler tool provides an easy and user friendly way to extract and analyze the epihaplotype composition of reads from targeted bisulfite sequencing experiments. ampliMethProfiler is written in python language and requires a local installation of BLAST and (optionally) QIIME tools. It can be run on Linux and OS X platforms. The software is open source and freely available at http://amplimethprofiler.sourceforge.net .

"Off-on" electrochemical hairpin-DNA-based genosensor for cancer diagnostics.

PubMed

Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt; Ferapontova, Elena E

2011-03-01

A simple and robust "off-on" signaling genosensor platform with improved selectivity for single-nucleotide polymorphism (SNP) detection based on the electronic DNA hairpin molecular beacons has been developed. The DNA beacons were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 3'-end, while the 5'-end was labeled with a methylene blue (MB) redox probe. A typical "on-off" change of the electrochemical signal was observed upon hybridization of the 27-33 nucleotide (nt) long hairpin DNA to the target DNA, in agreement with all the hitherto published data. Truncation of the DNA hairpin beacons down to 20 nts provided improved genosensor selectivity for SNP and allowed switching of the electrochemical genosensor response from the on-off to the off-on mode. Switching was consistent with the variation in the mechanism of the electron transfer reaction between the electrode and the MB redox label, for the folded beacon being characteristic of the electrochemistry of adsorbed species, while for the "open" duplex structure being formally controlled by the diffusion of the redox label within the adsorbate layer. The relative current intensities of both processes were governed by the length of the formed DNA duplex, potential scan rate, and apparent diffusion coefficient of the redox species. The off-on genosensor design used for detection of a cancer biomarker TP53 gene sequence favored discrimination between the healthy and SNP-containing DNA sequences, which was particularly pronounced at short hybridization times.

Chemiluminescent and chemiluminescence resonance energy transfer (CRET) detection of DNA, metal ions, and aptamer-substrate complexes using hemin/G-quadruplexes and CdSe/ZnS quantum dots.

PubMed

Freeman, Ronit; Liu, Xiaoqing; Willner, Itamar

2011-08-03

Nucleic acid subunits consisting of fragments of the horseradish peroxidase (HRP)-mimicking DNAzyme and aptamer domains against ATP or sequences recognizing Hg(2+) ions self-assemble, in the presence of ATP or Hg(2+), into the active hemin-G-quadruplex DNAzyme structure. The DNAzyme-generated chemiluminescence provides the optical readout for the sensing events. In addition, the DNAzyme-stimulated chemiluminescence resonance energy transfer (CRET) to CdSe/ZnS quantum dots (QDs) is implemented to develop aptamer or DNA sensing platforms. The self-assembly of the ATP-aptamer subunits/hemin-G-quadruplex DNAzyme, where one of the aptamer subunits is functionalized with CdSe/ZnS QDs, leads to the CRET signal. Also, the functionalization of QDs with a hairpin nucleic acid that includes the G-quadruplex sequence in a ''caged'' configuration is used to analyze DNA. The opening of the hairpin structure by the target DNA assembles the hemin-G-quadruplex DNAzyme that stimulates the CRET signal. By the application of three different sized QDs functionalized with different hairpins, the multiplexed analysis of three different DNA targets is demonstrated by the generation of three different CRET luminescence signals.

FBIS: A regional DNA barcode archival & analysis system for Indian fishes

PubMed Central

Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

2012-01-01

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. Availability The database is available for free at http://mail.nbfgr.res.in/fbis/ PMID:22715304

j5 v2.8.4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hillson, Nathan

j5 automates and optimizes the design of the molecular biological process of cloning/constructing DNA. j5 enables users to benefit from (combinatorial) multi-part scar-less SLIC, Gibson, CPEC, Golden Gate assembly, or variants thereof, for which automation software does not currently exist, without the intense labor currently associated with the process. j5 inputs a list of the DNA sequences to be assembled, along with a Genbank, FASTA, jbei-seq, or SBOL v1.1 format sequence file for each DNA source. Given the list of DNA sequences to be assembled, j5 first determines the cost-minimizing assembly strategy for each part (direct synthesis, PCR/SOE, or oligo-embedding),more » designs DNA oligos with Primer3, adds flanking homology sequences (SLIC, Gibson, and CPEC; optimized with Primer3 for CPEC) or optimized overhang sequences (Golden Gate) to the oligos and direct synthesis pieces, and utilizes BLAST to check against oligo mis-priming and assembly piece incompatibility events. After identifying DNA oligos that are already contained within a local collection for reuse, the program estimates the total cost of direct synthesis and new oligos to be ordered. In the instance that j5 identifies putative assembly piece incompatibilities (multiple pieces with high flanking sequence homology), the program suggests hierarchical subassemblies where possible. The program outputs a comma-separated value (CSV) file, viewable via Excel or other spreadsheet software, that contains assembly design information (such as the PCR/SOE reactions to perform, their anticipated sizes and sequences, etc.) as well as a properly annotated genbank file containing the sequence resulting from the assembly, and appends the local oligo library with the oligos to be ordered j5 condenses multiple independent assembly projects into 96-well format for high-throughput liquid-handling robotics platforms, and generates configuration files for the PR-PR biology-friendly robot programming language. j5 thus provides a new way to design DNA assembly procedures much more productively and efficiently, not only in terms of time, but also in terms of cost. To a large extent, however, j5 does not allow people to do something that could not be done before by hand given enough time and effort. An exception to this is that, since the very act of using j5 to design the DNA assembly process standardizes the experimental details and workflow, j5 enables a single person to concurrently perform the independent DNA construction tasks of an entire group of researchers. Currently, this is not readily possible, since separate researchers employ disparate design strategies and workflows, and furthermore, their designs and workflows are very infrequently fully captured in an electronic format which is conducive to automation.« less

Validation of next generation sequencing technologies in comparison to current diagnostic gold standards for BRAF, EGFR and KRAS mutational analysis.

PubMed

McCourt, Clare M; McArt, Darragh G; Mills, Ken; Catherwood, Mark A; Maxwell, Perry; Waugh, David J; Hamilton, Peter; O'Sullivan, Joe M; Salto-Tellez, Manuel

2013-01-01

Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis. Thirteen clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analysed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analysed using closed platform-specific proprietary bioinformatics software as well as open third party applications. Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology before consideration for diagnostic use.

Phylogenetic and Structural Analysis of the Pluripotency Factor Sex-Determining Region Y box2 Gene of Camelus dromedarius (cSox2).

PubMed

Alawad, Abdullah; Alharbi, Sultan; Alhazzaa, Othman; Alagrafi, Faisal; Alkhrayef, Mohammed; Alhamdan, Ziyad; Alenazi, Abdullah; Al-Johi, Hasan; Alanazi, Ibrahim O; Hammad, Mohamed

2016-01-01

Although the sequencing information of Sox2 cDNA for many mammalian is available, the Sox2 cDNA of Camelus dromedaries has not yet been characterized. The objective of this study was to sequence and characterize Sox2 cDNA from the brain of C. dromedarius (also known as Arabian camel). A full coding sequence of the Sox2 gene from the brain of C. dromedarius was amplified by reverse transcription PCRjmc and then sequenced using the 3730XL series platform Sequencer (Applied Biosystem) for the first time. The cDNA sequence displayed an open reading frame of 822 nucleotides, encoding a protein of 273 amino acids. The molecular weight and the isoelectric point of the translated protein were calculated as 29.825 kDa and 10.11, respectively, using bioinformatics analysis. The predicted cSox2 protein sequence exhibited high identity: 99% for Homo sapiens, Mus musculus, Bos taurus, and Vicugna pacos; 98% for Sus scrofa and 93% for Camelus ferus. A 3D structure was built based on the available crystal structure of the HMG-box domain of human stem cell transcription factor Sox2 (PDB: 2 LE4) with 81 residues and predicting bioinformatics software for 273 amino acid residues. The comparison confirms the presence of the HMG-box domain in the cSox2 protein. The orthologous phylogenetic analysis showed that the Sox2 isoform from C. dromedarius was grouped with humans, alpacas, cattle, and pigs. We believe that this genetic and structural information will be a helpful source for the annotation. Furthermore, Sox2 is one of the transcription factors that contributes to the generation-induced pluripotent stem cells (iPSCs), which in turn will probably help generate camel induced pluripotent stem cells (CiPSCs).

Rapid isolation of microsatellite DNAs and identification of polymorphic mitochondrial DNA regions in the fish rotan (Perccottus glenii) invading European Russia

USGS Publications Warehouse

King, Timothy L.; Eackles, Michael S.; Reshetnikov, Andrey N.

2015-01-01

Human-mediated translocations and subsequent large-scale colonization by the invasive fish rotan (Perccottus glenii Dybowski, 1877; Perciformes, Odontobutidae), also known as Amur or Chinese sleeper, has resulted in dramatic transformations of small lentic ecosystems. However, no detailed genetic information exists on population structure, levels of effective movement, or relatedness among geographic populations of P. glenii within the European part of the range. We used massively parallel genomic DNA shotgun sequencing on the semiconductor-based Ion Torrent Personal Genome Machine (PGM) sequencing platform to identify nuclear microsatellite and mitochondrial DNA sequences in P. glenii from European Russia. Here we describe the characterization of nine nuclear microsatellite loci, ascertain levels of allelic diversity, heterozygosity, and demographic status of P. glenii collected from Ilev, Russia, one of several initial introduction points in European Russia. In addition, we mapped sequence reads to the complete P. glenii mitochondrial DNA sequence to identify polymorphic regions. Nuclear microsatellite markers developed for P. glenii yielded sufficient genetic diversity to: (1) produce unique multilocus genotypes; (2) elucidate structure among geographic populations; and (3) provide unique perspectives for analysis of population sizes and historical demographics. Among 4.9 million filtered P. glenii Ion Torrent PGM sequence reads, 11,304 mapped to the mitochondrial genome (NC_020350). This resulted in 100 % coverage of this genome to a mean coverage depth of 102X. A total of 130 variable sites were observed between the publicly available genome from China and the studied composite mitochondrial genome. Among these, 82 were diagnostic and monomorphic between the mitochondrial genomes and distributed among 15 genome regions. The polymorphic sites (N = 48) were distributed among 11 mitochondrial genome regions. Our results also indicate that sequence reads generated from two three-hour runs on the Ion Torrent PGM can generate a sufficient number of nuclear and mitochondrial markers to improve understanding of the evolutionary and ecological dynamics of non-model and in particular, invasive species.

Deciphering the Epigenetic Code: An Overview of DNA Methylation Analysis Methods

PubMed Central

Umer, Muhammad

2013-01-01

Abstract Significance: Methylation of cytosine in DNA is linked with gene regulation, and this has profound implications in development, normal biology, and disease conditions in many eukaryotic organisms. A wide range of methods and approaches exist for its identification, quantification, and mapping within the genome. While the earliest approaches were nonspecific and were at best useful for quantification of total methylated cytosines in the chunk of DNA, this field has seen considerable progress and development over the past decades. Recent Advances: Methods for DNA methylation analysis differ in their coverage and sensitivity, and the method of choice depends on the intended application and desired level of information. Potential results include global methyl cytosine content, degree of methylation at specific loci, or genome-wide methylation maps. Introduction of more advanced approaches to DNA methylation analysis, such as microarray platforms and massively parallel sequencing, has brought us closer to unveiling the whole methylome. Critical Issues: Sensitive quantification of DNA methylation from degraded and minute quantities of DNA and high-throughput DNA methylation mapping of single cells still remain a challenge. Future Directions: Developments in DNA sequencing technologies as well as the methods for identification and mapping of 5-hydroxymethylcytosine are expected to augment our current understanding of epigenomics. Here we present an overview of methodologies available for DNA methylation analysis with special focus on recent developments in genome-wide and high-throughput methods. While the application focus relates to cancer research, the methods are equally relevant to broader issues of epigenetics and redox science in this special forum. Antioxid. Redox Signal. 18, 1972–1986. PMID:23121567

Real-time detection of BRAF V600E mutation from archival hairy cell leukemia FFPE tissue by nanopore sequencing.

PubMed

Vacca, Davide; Cancila, Valeria; Gulino, Alessandro; Lo Bosco, Giosuè; Belmonte, Beatrice; Di Napoli, Arianna; Florena, Ada Maria; Tripodo, Claudio; Arancio, Walter

2018-02-01

The MinION is a miniaturized high-throughput next generation sequencing platform of novel conception. The use of nucleic acids derived from formalin-fixed paraffin-embedded samples is highly desirable, but their adoption for molecular assays is hurdled by the high degree of fragmentation and by the chemical-induced mutations stemming from the fixation protocols. In order to investigate the suitability of MinION sequencing on formalin-fixed paraffin-embedded samples, the presence and frequency of BRAF c.1799T > A mutation was investigated in two archival tissue specimens of Hairy cell leukemia and Hairy cell leukemia Variant. Despite the poor quality of the starting DNA, BRAF mutation was successfully detected in the Hairy cell leukemia sample with around 50% of the reads obtained within 2 h of the sequencing start. Notably, the mutational burden of the Hairy cell leukemia sample as derived from nanopore sequencing proved to be comparable to a sensitive method for the detection of point mutations, namely the Digital PCR, using a validated assay. Nanopore sequencing can be adopted for targeted sequencing of genetic lesions on critical DNA samples such as those extracted from archival routine formalin-fixed paraffin-embedded samples. This result let speculating about the possibility that the nanopore sequencing could be trustably adopted for the real-time targeted sequencing of genetic lesions. Our report opens the window for the adoption of nanopore sequencing in molecular pathology for research and diagnostics.

High-throughput sequencing of fecal DNA to identify insects consumed by wild Weddell's saddleback tamarins (Saguinus weddelli, Cebidae, Primates) in Bolivia.

PubMed

Mallott, E K; Malhi, R S; Garber, P A

2015-03-01

The genus Saguinus represents a successful radiation of over 20 species of small-bodied New World monkeys. Studies of the tamarin diet indicate that insects and small vertebrates account for ∼16-45% of total feeding and foraging time, and represent an important source of lipids, protein, and metabolizable energy. Although tamarins are reported to commonly consume large-bodied insects such as grasshoppers and walking sticks (Orthoptera), little is known concerning the degree to which smaller or less easily identifiable arthropod prey comprises an important component of their diet. To better understand tamarin arthropod feeding behavior, fecal samples from 20 wild Bolivian saddleback tamarins (members of five groups) were collected over a 3 week period in June 2012, and analyzed for the presence of arthropod DNA. DNA was extracted using a Qiagen stool extraction kit, and universal insect primers were created and used to amplify a ∼280 bp section of the COI mitochondrial gene. Amplicons were sequenced on the Roche 454 sequencing platform using high-throughput sequencing techniques. An analysis of these samples indicated the presence of 43 taxa of arthropods including 10 orders, 15 families, and 12 identified genera. Many of these taxa had not been previously identified in the tamarin diet. These results highlight molecular analysis of fecal DNA as an important research tool for identifying anthropod feeding patterns in primates, and reveal broad diversity in the taxa, foraging microhabitats, and size of arthropods consumed by tamarin monkeys. © 2014 Wiley Periodicals, Inc.

EST-PAC a web package for EST annotation and protein sequence prediction

PubMed Central

Strahm, Yvan; Powell, David; Lefèvre, Christophe

2006-01-01

With the decreasing cost of DNA sequencing technology and the vast diversity of biological resources, researchers increasingly face the basic challenge of annotating a larger number of expressed sequences tags (EST) from a variety of species. This typically consists of a series of repetitive tasks, which should be automated and easy to use. The results of these annotation tasks need to be stored and organized in a consistent way. All these operations should be self-installing, platform independent, easy to customize and amenable to using distributed bioinformatics resources available on the Internet. In order to address these issues, we present EST-PAC a web oriented multi-platform software package for expressed sequences tag (EST) annotation. EST-PAC provides a solution for the administration of EST and protein sequence annotations accessible through a web interface. Three aspects of EST annotation are automated: 1) searching local or remote biological databases for sequence similarities using Blast services, 2) predicting protein coding sequence from EST data and, 3) annotating predicted protein sequences with functional domain predictions. In practice, EST-PAC integrates the BLASTALL suite, EST-Scan2 and HMMER in a relational database system accessible through a simple web interface. EST-PAC also takes advantage of the relational database to allow consistent storage, powerful queries of results and, management of the annotation process. The system allows users to customize annotation strategies and provides an open-source data-management environment for research and education in bioinformatics. PMID:17147782

Review of sequencing platforms and their applications in phaeochromocytoma and paragangliomas.

PubMed

Pillai, Suja; Gopalan, Vinod; Lam, Alfred King-Yin

2017-08-01

Genetic testing is recommended for patients with phaeochromocytoma (PCC) and paraganglioma (PGL) because of their genetic heterogeneity and heritability. Due to the large number of susceptibility genes associated with PCC/PGL, next-generation sequencing (NGS) technology is ideally suited for carrying out genetic screening of these individuals. New generations of DNA sequencing technologies facilitate the development of comprehensive genetic testing in PCC/PGL at a lower cost. Whole-exome sequencing and targeted NGS are the preferred methods for screening of PCC/PGL, both having precise mutation detection methods and low costs. RNA sequencing and DNA methylation studies using NGS technology in PCC/PGL can be adopted to act as diagnostic or prognostic biomarkers as well as in planning targeted epigenetic treatment of patients with PCC/PGL. The designs of NGS having a high depth of coverage and robust analytical pipelines can lead to the successful detection of a wide range of genomic defects in PCC/PGL. Nevertheless, the major challenges of this technology must be addressed before it has practical applications in the clinical diagnostics to fulfill the goal of personalized medicine in PCC/PGL. In future, novel approaches of sequencing, such as third and fourth generation sequencing can alter the workflow, cost, analysis, and interpretation of genomics associated with PCC/PGL. Copyright © 2017 Elsevier B.V. All rights reserved.

ScaffoldSeq: Software for characterization of directed evolution populations.

PubMed

Woldring, Daniel R; Holec, Patrick V; Hackel, Benjamin J

2016-07-01

ScaffoldSeq is software designed for the numerous applications-including directed evolution analysis-in which a user generates a population of DNA sequences encoding for partially diverse proteins with related functions and would like to characterize the single site and pairwise amino acid frequencies across the population. A common scenario for enzyme maturation, antibody screening, and alternative scaffold engineering involves naïve and evolved populations that contain diversified regions, varying in both sequence and length, within a conserved framework. Analyzing the diversified regions of such populations is facilitated by high-throughput sequencing platforms; however, length variability within these regions (e.g., antibody CDRs) encumbers the alignment process. To overcome this challenge, the ScaffoldSeq algorithm takes advantage of conserved framework sequences to quickly identify diverse regions. Beyond this, unintended biases in sequence frequency are generated throughout the experimental workflow required to evolve and isolate clones of interest prior to DNA sequencing. ScaffoldSeq software uniquely handles this issue by providing tools to quantify and remove background sequences, cluster similar protein families, and dampen the impact of dominant clones. The software produces graphical and tabular summaries for each region of interest, allowing users to evaluate diversity in a site-specific manner as well as identify epistatic pairwise interactions. The code and detailed information are freely available at http://research.cems.umn.edu/hackel. Proteins 2016; 84:869-874. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.

PubMed

Rousseau, Beth A; Hou, Zhonggang; Gramelspacher, Max J; Zhang, Yan

2018-03-01

The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavage can be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA. Copyright © 2018 Elsevier Inc. All rights reserved.

Cell-Free Expression and In Situ Immobilization of Parasite Proteins from Clonorchis sinensis for Rapid Identification of Antigenic Candidates

PubMed Central

Ju, Jung Won; Kim, Ho-Cheol; Shin, Hyun-Il; Kim, Yu Jung; Kim, Dong-Myung

2015-01-01

Progress towards genetic sequencing of human parasites has provided the groundwork for a post-genomic approach to develop novel antigens for the diagnosis and treatment of parasite infections. To fully utilize the genomic data, however, high-throughput methodologies are required for functional analysis of the proteins encoded in the genomic sequences. In this study, we investigated cell-free expression and in situ immobilization of parasite proteins as a novel platform for the discovery of antigenic proteins. PCR-amplified parasite DNA was immobilized on microbeads that were also functionalized to capture synthesized proteins. When the microbeads were incubated in a reaction mixture for cell-free synthesis, proteins expressed from the microbead-immobilized DNA were instantly immobilized on the same microbeads, providing a physical linkage between the genetic information and encoded proteins. This approach of in situ expression and isolation enables streamlined recovery and analysis of cell-free synthesized proteins and also allows facile identification of the genes coding antigenic proteins through direct PCR of the microbead-bound DNA. PMID:26599101

High sensitive and direct fluorescence detection of single viral DNA sequences by integration of double strand probes onto microgels particles.

PubMed

Aliberti, A; Cusano, A M; Battista, E; Causa, F; Netti, P A

2016-02-21

A novel class of probes for fluorescence detection was developed and combined to microgel particles for a high sensitive fluorescence detection of nucleic acids. A double strand probe with an optimized fluorescent-quencher couple was designed for the detection of different lengths of nucleic acids (39 nt and 100 nt). Such probe proved efficient in target detection in different contests and specific even in presence of serum proteins. The conjugation of double strand probes onto polymeric microgels allows for a sensitive detection of DNA sequences from HIV, HCV and SARS corona viruses with a LOD of 1.4 fM, 3.7 fM and 1.4 fM, respectively, and with a dynamic range of 10(-9)-10(-15) M. Such combination enhances the sensitivity of the detection of almost five orders of magnitude when compared to the only probe. The proposed platform based on the integration of innovative double strand probe into microgels particles represents an attractive alternative to conventional sensitive DNA detection technologies that rely on amplifications methods.

Identification of Biomolecular Building Blocks by Recognition Tunneling: Stride towards Nanopore Sequencing of Biomolecules

NASA Astrophysics Data System (ADS)

Sen, Suman

DNA, RNA and Protein are three pivotal biomolecules in human and other organisms, playing decisive roles in functionality, appearance, diseases development and other physiological phenomena. Hence, sequencing of these biomolecules acquires the prime interest in the scientific community. Single molecular identification of their building blocks can be done by a technique called Recognition Tunneling (RT) based on Scanning Tunneling Microscope (STM). A single layer of specially designed recognition molecule is attached to the STM electrodes, which trap the targeted molecules (DNA nucleoside monophosphates, RNA nucleoside monophosphates or amino acids) inside the STM nanogap. Depending on their different binding interactions with the recognition molecules, the analyte molecules generate stochastic signal trains accommodating their "electronic fingerprints". Signal features are used to detect the molecules using a machine learning algorithm and different molecules can be identified with significantly high accuracy. This, in turn, paves the way for rapid, economical nanopore sequencing platform, overcoming the drawbacks of Next Generation Sequencing (NGS) techniques. To read DNA nucleotides with high accuracy in an STM tunnel junction a series of nitrogen-based heterocycles were designed and examined to check their capabilities to interact with naturally occurring DNA nucleotides by hydrogen bonding in the tunnel junction. These recognition molecules are Benzimidazole, Imidazole, Triazole and Pyrrole. Benzimidazole proved to be best among them showing DNA nucleotide classification accuracy close to 99%. Also, Imidazole reader can read an abasic monophosphate (AP), a product from depurination or depyrimidination that occurs 10,000 times per human cell per day. In another study, I have investigated a new universal reader, 1-(2-mercaptoethyl)pyrene (Pyrene reader) based on stacking interactions, which should be more specific to the canonical DNA nucleosides. In addition, Pyrene reader showed higher DNA base-calling accuracy compare to Imidazole reader, the workhorse in our previous projects. In my other projects, various amino acids and RNA nucleoside monophosphates were also classified with significantly high accuracy using RT. Twenty naturally occurring amino acids and various RNA nucleosides (four canonical and two modified) were successfully identified. Thus, we envision nanopore sequencing biomolecules using Recognition Tunneling (RT) that should provide comprehensive betterment over current technologies in terms of time, chemical and instrumental cost and capability of de novo sequencing.

JNSViewer—A JavaScript-based Nucleotide Sequence Viewer for DNA/RNA secondary structures

PubMed Central

Dong, Min; Graham, Mitchell; Yadav, Nehul

2017-01-01

Many tools are available for visualizing RNA or DNA secondary structures, but there is scarce implementation in JavaScript that provides seamless integration with the increasingly popular web computational platforms. We have developed JNSViewer, a highly interactive web service, which is bundled with several popular tools for DNA/RNA secondary structure prediction and can provide precise and interactive correspondence among nucleotides, dot-bracket data, secondary structure graphs, and genic annotations. In JNSViewer, users can perform RNA secondary structure predictions with different programs and settings, add customized genic annotations in GFF format to structure graphs, search for specific linear motifs, and extract relevant structure graphs of sub-sequences. JNSViewer also allows users to choose a transcript or specific segment of Arabidopsis thaliana genome sequences and predict the corresponding secondary structure. Popular genome browsers (i.e., JBrowse and BrowserGenome) were integrated into JNSViewer to provide powerful visualizations of chromosomal locations, genic annotations, and secondary structures. In addition, we used StructureFold with default settings to predict some RNA structures for Arabidopsis by incorporating in vivo high-throughput RNA structure profiling data and stored the results in our web server, which might be a useful resource for RNA secondary structure studies in plants. JNSViewer is available at http://bioinfolab.miamioh.edu/jnsviewer/index.html. PMID:28582416

Homogeneous assay of target molecules based on chemiluminescence resonance energy transfer (CRET) using DNAzyme-linked aptamers.

PubMed

Mun, Hyoyoung; Jo, Eun-Jung; Li, Taihua; Joung, Hyou-Arm; Hong, Dong-Gu; Shim, Won-Bo; Jung, Cheulhee; Kim, Min-Gon

2014-08-15

We have designed a single-stranded DNAzyme-aptamer sensor for homogeneous target molecular detection based on chemiluminescence resonance energy transfer (CRET). The structure of the engineered single-stranded DNA (ssDNA) includes the horseradish peroxidase (HRP)-like DNAzyme, optimum-length linker (10-mer-length DNA), and target-specific aptamer sequences. A quencher dye was modified at the 3' end of the aptamer sequence. The incorporation of hemin into the G-quadruplex structure of DNAzyme yields an active HRP-like activity that catalyzes luminol to generate a chemiluminescence (CL) signal. In the presence of target molecules, such as ochratoxin A (OTA), adenosine triphosphate (ATP), or thrombin, the aptamer sequence was folded due to the formation of the aptamer/analyte complex, which induced the quencher dye close to the DNAzyme structure. Consequently, the CRET occurred between a DNAzyme-catalyzed chemiluminescence reaction and the quencher dye. Our results showed that CRET-based DNAzyme-aptamer biosensing enabled specific OTA analysis with a limit of detection of 0.27ng/mL. The CRET platform needs no external light source and avoids autofluorescence and photobleaching, and target molecules can be detected specifically and sensitively in a homogeneous manner. Copyright © 2014 Elsevier B.V. All rights reserved.

Whole-Genome Sequencing in Microbial Forensic Analysis of Gamma-Irradiated Microbial Materials.

PubMed

Broomall, Stacey M; Ait Ichou, Mohamed; Krepps, Michael D; Johnsky, Lauren A; Karavis, Mark A; Hubbard, Kyle S; Insalaco, Joseph M; Betters, Janet L; Redmond, Brady W; Rivers, Bryan A; Liem, Alvin T; Hill, Jessica M; Fochler, Edward T; Roth, Pierce A; Rosenzweig, C Nicole; Skowronski, Evan W; Gibbons, Henry S

2016-01-15

Effective microbial forensic analysis of materials used in a potential biological attack requires robust methods of morphological and genetic characterization of the attack materials in order to enable the attribution of the materials to potential sources and to exclude other potential sources. The genetic homogeneity and potential intersample variability of many of the category A to C bioterrorism agents offer a particular challenge to the generation of attributive signatures, potentially requiring whole-genome or proteomic approaches to be utilized. Currently, irradiation of mail is standard practice at several government facilities judged to be at particularly high risk. Thus, initial forensic signatures would need to be recovered from inactivated (nonviable) material. In the study described in this report, we determined the effects of high-dose gamma irradiation on forensic markers of bacterial biothreat agent surrogate organisms with a particular emphasis on the suitability of genomic DNA (gDNA) recovered from such sources as a template for whole-genome analysis. While irradiation of spores and vegetative cells affected the retention of Gram and spore stains and sheared gDNA into small fragments, we found that irradiated material could be utilized to generate accurate whole-genome sequence data on the Illumina and Roche 454 sequencing platforms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Droplet-Based Pyrosequencing Using Digital Microfluidics

PubMed Central

Boles, Deborah J.; Benton, Jonathan L.; Siew, Germaine J.; Levy, Miriam H.; Thwar, Prasanna K.; Sandahl, Melissa A.; Rouse, Jeremy L.; Perkins, Lisa C.; Sudarsan, Arjun P.; Jalili, Roxana; Pamula, Vamsee K.; Srinivasan, Vijay; Fair, Richard B.; Griffin, Peter B.; Eckhardt, Allen E.; Pollack, Michael G.

2013-01-01

The feasibility of implementing pyrosequencing chemistry within droplets using electrowetting-based digital microfluidics is reported. An array of electrodes patterned on a printed-circuit board was used to control the formation, transportation, merging, mixing, and splitting of submicroliter-sized droplets contained within an oil-filled chamber. A three-enzyme pyrosequencing protocol was implemented in which individual droplets contained enzymes, deoxyribonucleotide triphosphates (dNTPs), and DNA templates. The DNA templates were anchored to magnetic beads which enabled them to be thoroughly washed between nucleotide additions. Reagents and protocols were optimized to maximize signal over background, linearity of response, cycle efficiency, and wash efficiency. As an initial demonstration of feasibility, a portion of a 229 bp Candida parapsilosis template was sequenced using both a de novo protocol and a resequencing protocol. The resequencing protocol generated over 60 bp of sequence with 100% sequence accuracy based on raw pyrogram levels. Excellent linearity was observed for all of the homopolymers (two, three, or four nucleotides) contained in the C. parapsilosis sequence. With improvements in microfluidic design it is expected that longer reads, higher throughput, and improved process integration (i.e., “sample-to-sequence” capability) could eventually be achieved using this low-cost platform. PMID:21932784

Next stop for the CRISPR revolution: RNA-guided epigenetic regulators.

PubMed

Vora, Suhani; Tuttle, Marcelle; Cheng, Jenny; Church, George

2016-09-01

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins offer a breakthrough platform for cheap, programmable, and effective sequence-specific DNA targeting. The CRISPR-Cas system is naturally equipped for targeted DNA cutting through its native nuclease activity. As such, groups researching a broad spectrum of biological organisms have quickly adopted the technology with groundbreaking applications to genomic sequence editing in over 20 different species. However, the biological code of life is not only encoded in genetics but also in epigenetics as well. While genetic sequence editing is a powerful ability, we must also be able to edit and regulate transcriptional and epigenetic code. Taking inspiration from work on earlier sequence-specific targeting technologies such as zinc fingers (ZFs) and transcription activator-like effectors (TALEs), researchers quickly expanded the CRISPR-Cas toolbox to include transcriptional activation, repression, and epigenetic modification. In this review, we highlight advances that extend the CRISPR-Cas toolkit for transcriptional and epigenetic regulation, as well as best practice guidelines for these tools, and a perspective on future applications. © 2016 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Introducing W.A.T.E.R.S.: a workflow for the alignment, taxonomy, and ecology of ribosomal sequences.

PubMed

Hartman, Amber L; Riddle, Sean; McPhillips, Timothy; Ludäscher, Bertram; Eisen, Jonathan A

2010-06-12

For more than two decades microbiologists have used a highly conserved microbial gene as a phylogenetic marker for bacteria and archaea. The small-subunit ribosomal RNA gene, also known as 16 S rRNA, is encoded by ribosomal DNA, 16 S rDNA, and has provided a powerful comparative tool to microbial ecologists. Over time, the microbial ecology field has matured from small-scale studies in a select number of environments to massive collections of sequence data that are paired with dozens of corresponding collection variables. As the complexity of data and tool sets have grown, the need for flexible automation and maintenance of the core processes of 16 S rDNA sequence analysis has increased correspondingly. We present WATERS, an integrated approach for 16 S rDNA analysis that bundles a suite of publicly available 16 S rDNA analysis software tools into a single software package. The "toolkit" includes sequence alignment, chimera removal, OTU determination, taxonomy assignment, phylogentic tree construction as well as a host of ecological analysis and visualization tools. WATERS employs a flexible, collection-oriented 'workflow' approach using the open-source Kepler system as a platform. By packaging available software tools into a single automated workflow, WATERS simplifies 16 S rDNA analyses, especially for those without specialized bioinformatics, programming expertise. In addition, WATERS, like some of the newer comprehensive rRNA analysis tools, allows researchers to minimize the time dedicated to carrying out tedious informatics steps and to focus their attention instead on the biological interpretation of the results. One advantage of WATERS over other comprehensive tools is that the use of the Kepler workflow system facilitates result interpretation and reproducibility via a data provenance sub-system. Furthermore, new "actors" can be added to the workflow as desired and we see WATERS as an initial seed for a sizeable and growing repository of interoperable, easy-to-combine tools for asking increasingly complex microbial ecology questions.

Programmable autonomous synthesis of single-stranded DNA

NASA Astrophysics Data System (ADS)

Kishi, Jocelyn Y.; Schaus, Thomas E.; Gopalkrishnan, Nikhil; Xuan, Feng; Yin, Peng

2018-02-01

DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

Programmable autonomous synthesis of single-stranded DNA.

PubMed

Kishi, Jocelyn Y; Schaus, Thomas E; Gopalkrishnan, Nikhil; Xuan, Feng; Yin, Peng

2018-02-01

DNA performs diverse functional roles in biology, nanotechnology and biotechnology, but current methods for autonomously synthesizing arbitrary single-stranded DNA are limited. Here, we introduce the concept of primer exchange reaction (PER) cascades, which grow nascent single-stranded DNA with user-specified sequences following prescribed reaction pathways. PER synthesis happens in a programmable, autonomous, in situ and environmentally responsive fashion, providing a platform for engineering molecular circuits and devices with a wide range of sensing, monitoring, recording, signal-processing and actuation capabilities. We experimentally demonstrate a nanodevice that transduces the detection of a trigger RNA into the production of a DNAzyme that degrades an independent RNA substrate, a signal amplifier that conditionally synthesizes long fluorescent strands only in the presence of a particular RNA signal, molecular computing circuits that evaluate logic (AND, OR, NOT) combinations of RNA inputs, and a temporal molecular event recorder that records in the PER transcript the order in which distinct RNA inputs are sequentially detected.

Single-Molecule Denaturation Mapping of DNA in Nanofluidic Channels

NASA Astrophysics Data System (ADS)

Reisner, Walter; Larsen, Niels; Silahtaroglu, Asli; Kristensen, Anders; Tommerup, Niels; Tegenfeldt, Jonas O.; Flyvbjerg, Henrik

2010-03-01

Nanochannel based DNA stretching can serve as a platform for a new optical mapping technique based on measuring the pattern of partial melting along the extended molecules. We partially melt DNA extended in nanofluidic channels via a combination of local heating and added chemical denaturants. The melted molecules, imaged via a standard fluorescence videomicroscopy setup, exhibit a nonuniform fluorescence profile corresponding to a series of local dips and peaks in the intensity trace along the stretched molecule. We show that this barcode is consistent with the presence of locally melted regions along the molecule and can be explained by calculations of sequence-dependent melting probability. Specifically, we obtain experimental melting profiles for T4, T7, lambda-phage and bacterial artificial chromosome DNA (from human chromosome 12) and compare these profiles to theory. In addition, we demonstrate that the BAC melting profile can be used to align the BAC to its correct position on chromosome 12.

VIP Barcoding: composition vector-based software for rapid species identification based on DNA barcoding.

PubMed

Fan, Long; Hui, Jerome H L; Yu, Zu Guo; Chu, Ka Hou

2014-07-01

Species identification based on short sequences of DNA markers, that is, DNA barcoding, has emerged as an integral part of modern taxonomy. However, software for the analysis of large and multilocus barcoding data sets is scarce. The Basic Local Alignment Search Tool (BLAST) is currently the fastest tool capable of handling large databases (e.g. >5000 sequences), but its accuracy is a concern and has been criticized for its local optimization. However, current more accurate software requires sequence alignment or complex calculations, which are time-consuming when dealing with large data sets during data preprocessing or during the search stage. Therefore, it is imperative to develop a practical program for both accurate and scalable species identification for DNA barcoding. In this context, we present VIP Barcoding: a user-friendly software in graphical user interface for rapid DNA barcoding. It adopts a hybrid, two-stage algorithm. First, an alignment-free composition vector (CV) method is utilized to reduce searching space by screening a reference database. The alignment-based K2P distance nearest-neighbour method is then employed to analyse the smaller data set generated in the first stage. In comparison with other software, we demonstrate that VIP Barcoding has (i) higher accuracy than Blastn and several alignment-free methods and (ii) higher scalability than alignment-based distance methods and character-based methods. These results suggest that this platform is able to deal with both large-scale and multilocus barcoding data with accuracy and can contribute to DNA barcoding for modern taxonomy. VIP Barcoding is free and available at http://msl.sls.cuhk.edu.hk/vipbarcoding/. © 2014 John Wiley & Sons Ltd.

MethSMRT: an integrative database for DNA N6-methyladenine and N4-methylcytosine generated by single-molecular real-time sequencing.

PubMed

Ye, Pohao; Luan, Yizhao; Chen, Kaining; Liu, Yizhi; Xiao, Chuanle; Xie, Zhi

2017-01-04

DNA methylation is an important type of epigenetic modifications, where 5- methylcytosine (5mC), 6-methyadenine (6mA) and 4-methylcytosine (4mC) are the most common types. Previous efforts have been largely focused on 5mC, providing invaluable insights into epigenetic regulation through DNA methylation. Recently developed single-molecule real-time (SMRT) sequencing technology provides a unique opportunity to detect the less studied DNA 6mA and 4mC modifications at single-nucleotide resolution. With a rapidly increased amount of SMRT sequencing data generated, there is an emerging demand to systematically explore DNA 6mA and 4mC modifications from these data sets. MethSMRT is the first resource hosting DNA 6mA and 4mC methylomes. All the data sets were processed using the same analysis pipeline with the same quality control. The current version of the database provides a platform to store, browse, search and download epigenome-wide methylation profiles of 156 species, including seven eukaryotes such as Arabidopsis, C. elegans, Drosophila, mouse and yeast, as well as 149 prokaryotes. It also offers a genome browser to visualize the methylation sites and related information such as single nucleotide polymorphisms (SNP) and genomic annotation. Furthermore, the database provides a quick summary of statistics of methylome of 6mA and 4mC and predicted methylation motifs for each species. MethSMRT is publicly available at http://sysbio.sysu.edu.cn/methsmrt/ without use restriction. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Detection of single-nucleotide polymorphisms using an ON-OFF switching of regenerated biosensor based on a locked nucleic acid-integrated and toehold-mediated strand displacement reaction.

PubMed

Gao, Zhong Feng; Ling, Yu; Lu, Lu; Chen, Ning Yu; Luo, Hong Qun; Li, Nian Bing

2014-03-04

Although various strategies have been reported for single-nucleotide polymorphisms (SNPs) detection, development of a time-saving, specific, and regenerated electrochemical sensing platform still remains a realistic goal. In this study, an ON-OFF switching of a regenerated biosensor based on a locked nucleic acid (LNA)-integrated and toehold-mediated strand displacement reaction technique is constructed for detection of SNPs. The LNA-integrated and methylene blue-labeled capture probe with an external toehold is designed to switch on the sensing system. The mutant-type DNA probe completes complementary with the capture probe to trigger the strand displacement reaction, which switches off the sensing system. However, when the single-base mismatched wild-type DNA probe is presented, the strand displacement reaction cannot be achieved; therefore, the sensing system still keeps the ON state. This DNA sensor is stable over five reuses. We further testify that the LNA-integrated sequence has better recognition ability for SNPs detection compared to the DNA-integrated sequence. Moreover, this DNA senor exhibits a remarkable discrimination capability of SNPs among abundant wild-type targets and 6000-fold (m/m) excess of genomic DNA. In addition, it is selective enough in complex and contaminant-ridden samples, such as human urine, soil, saliva, and beer. Overall, these results demonstrate that this reliable DNA sensor is easy to be fabricated, simple to operate, and stable enough to be readily regenerated.

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

PubMed Central

2012-01-01

Background In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. Results Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program. Conclusions We demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species. PMID:22805587

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

PubMed

Yang, Huaan; Tao, Ye; Zheng, Zequn; Li, Chengdao; Sweetingham, Mark W; Howieson, John G

2012-07-17

In the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding. Twenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program. We demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species.

Proteome Studies of Filamentous Fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Baker, Scott E.; Panisko, Ellen A.

2011-04-20

The continued fast pace of fungal genome sequence generation has enabled proteomic analysis of a wide breadth of organisms that span the breadth of the Kingdom Fungi. There is some phylogenetic bias to the current catalog of fungi with reasonable DNA sequence databases (genomic or EST) that could be analyzed at a global proteomic level. However, the rapid development of next generation sequencing platforms has lowered the cost of genome sequencing such that in the near future, having a genome sequence will no longer be a time or cost bottleneck for downstream proteomic (and transcriptomic) analyses. High throughput, non-gel basedmore » proteomics offers a snapshot of proteins present in a given sample at a single point in time. There are a number of different variations on the general method and technologies for identifying peptides in a given sample. We present a method that can serve as a “baseline” for proteomic studies of fungi.« less

Proteome studies of filamentous fungi.

PubMed

Baker, Scott E; Panisko, Ellen A

2011-01-01

The continued fast pace of fungal genome sequence generation has enabled proteomic analysis of a wide variety of organisms that span the breadth of the Kingdom Fungi. There is some phylogenetic bias to the current catalog of fungi with reasonable DNA sequence databases (genomic or EST) that could be analyzed at a global proteomic level. However, the rapid development of next generation sequencing platforms has lowered the cost of genome sequencing such that in the near future, having a genome sequence will no longer be a time or cost bottleneck for downstream proteomic (and transcriptomic) analyses. High throughput, nongel-based proteomics offers a snapshot of proteins present in a given sample at a single point in time. There are a number of variations on the general methods and technologies for identifying peptides in a given sample. We present a method that can serve as a "baseline" for proteomic studies of fungi.

CRISPR/Cas9 in Genome Editing and Beyond.

PubMed

Wang, Haifeng; La Russa, Marie; Qi, Lei S

2016-06-02

The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

Improvement in the amine glass platform by bubbling method for a DNA microarray

PubMed Central

Jee, Seung Hyun; Kim, Jong Won; Lee, Ji Hyeong; Yoon, Young Soo

2015-01-01

A glass platform with high sensitivity for sexually transmitted diseases microarray is described here. An amino-silane-based self-assembled monolayer was coated on the surface of a glass platform using a novel bubbling method. The optimized surface of the glass platform had highly uniform surface modifications using this method, as well as improved hybridization properties with capture probes in the DNA microarray. On the basis of these results, the improved glass platform serves as a highly reliable and optimal material for the DNA microarray. Moreover, in this study, we demonstrated that our glass platform, manufactured by utilizing the bubbling method, had higher uniformity, shorter processing time, lower background signal, and higher spot signal than the platforms manufactured by the general dipping method. The DNA microarray manufactured with a glass platform prepared using bubbling method can be used as a clinical diagnostic tool. PMID:26468293

Improvement in the amine glass platform by bubbling method for a DNA microarray.

PubMed

Jee, Seung Hyun; Kim, Jong Won; Lee, Ji Hyeong; Yoon, Young Soo

2015-01-01

A glass platform with high sensitivity for sexually transmitted diseases microarray is described here. An amino-silane-based self-assembled monolayer was coated on the surface of a glass platform using a novel bubbling method. The optimized surface of the glass platform had highly uniform surface modifications using this method, as well as improved hybridization properties with capture probes in the DNA microarray. On the basis of these results, the improved glass platform serves as a highly reliable and optimal material for the DNA microarray. Moreover, in this study, we demonstrated that our glass platform, manufactured by utilizing the bubbling method, had higher uniformity, shorter processing time, lower background signal, and higher spot signal than the platforms manufactured by the general dipping method. The DNA microarray manufactured with a glass platform prepared using bubbling method can be used as a clinical diagnostic tool.

Quantifying environmental DNA signals for aquatic invasive species across multiple detection platforms.

PubMed

Nathan, Lucas M; Simmons, Megan; Wegleitner, Benjamin J; Jerde, Christopher L; Mahon, Andrew R

2014-11-04

The use of molecular surveillance techniques has become popular among aquatic researchers and managers due to the improved sensitivity and efficiency compared to traditional sampling methods. Rapid expansion in the use of environmental DNA (eDNA), paired with the advancement of molecular technologies, has resulted in new detection platforms and techniques. In this study we present a comparison of three eDNA surveillance platforms: traditional polymerase chain reaction (PCR), quantitative PCR (qPCR), and digital droplet PCR (ddPCR) in which water samples were collected over a 24 h time period from mesocosm experiments containing a population gradient of invasive species densities. All platforms reliably detected the presence of DNA, even at low target organism densities within the first hour. The two quantitative platforms (qPCR and ddPCR) produced similar estimates of DNA concentrations. The analyses completed with ddPCR was faster from sample collection through analyses and cost approximately half the expenditure of qPCR. Although a new platform for eDNA surveillance of aquatic species, ddPCR was consistent with more commonly used qPCR and a cost-effective means of estimating DNA concentrations. Use of ddPCR by researchers and managers should be considered in future eDNA surveillance applications.

A statistical method for the detection of variants from next-generation resequencing of DNA pools.

PubMed

Bansal, Vikas

2010-06-15

Next-generation sequencing technologies have enabled the sequencing of several human genomes in their entirety. However, the routine resequencing of complete genomes remains infeasible. The massive capacity of next-generation sequencers can be harnessed for sequencing specific genomic regions in hundreds to thousands of individuals. Sequencing-based association studies are currently limited by the low level of multiplexing offered by sequencing platforms. Pooled sequencing represents a cost-effective approach for studying rare variants in large populations. To utilize the power of DNA pooling, it is important to accurately identify sequence variants from pooled sequencing data. Detection of rare variants from pooled sequencing represents a different challenge than detection of variants from individual sequencing. We describe a novel statistical approach, CRISP [Comprehensive Read analysis for Identification of Single Nucleotide Polymorphisms (SNPs) from Pooled sequencing] that is able to identify both rare and common variants by using two approaches: (i) comparing the distribution of allele counts across multiple pools using contingency tables and (ii) evaluating the probability of observing multiple non-reference base calls due to sequencing errors alone. Information about the distribution of reads between the forward and reverse strands and the size of the pools is also incorporated within this framework to filter out false variants. Validation of CRISP on two separate pooled sequencing datasets generated using the Illumina Genome Analyzer demonstrates that it can detect 80-85% of SNPs identified using individual sequencing while achieving a low false discovery rate (3-5%). Comparison with previous methods for pooled SNP detection demonstrates the significantly lower false positive and false negative rates for CRISP. Implementation of this method is available at http://polymorphism.scripps.edu/~vbansal/software/CRISP/.

A Sensitive DNA Capacitive Biosensor Using Interdigitated Electrodes

PubMed Central

Wang, Lei; Veselinovic, Milena; Yang, Lang; Geiss, Brian J.; Dandy, David S.; Chen, Tom

2017-01-01

This paper presents a label-free affinity-based capacitive biosensor using interdigitated electrodes. Using an optimized process of DNA probe preparation to minimize the effect of contaminants in commercial thiolated DNA probe, the electrode surface was functionalized with the 24-nucleotide DNA probes based on the West Nile virus sequence (Kunjin strain). The biosensor has the ability to detect complementary DNA fragments with a detection limit down to 20 DNA target molecules (1.5 aM range), making it suitable for a practical point-of-care (POC) platform for low target count clinical applications without the need for amplification. The reproducibility of the biosensor detection was improved with efficient covalent immobilization of purified single-stranded DNA probe oligomers on cleaned gold microelectrodes. In addition to the low detection limit, the biosensor showed a dynamic range of detection from 1 μL−1 to 105 μL−1 target molecules (20 to 2 million targets), making it suitable for sample analysis in a typical clinical application environment. The binding results presented in this paper were validated using fluorescent oligomers. PMID:27619528

Phylogeography of the Qinghai-Tibetan Plateau endemic Juniperus przewalskii (Cupressaceae) inferred from chloroplast DNA sequence variation.

PubMed

Zhang, Q; Chiang, T Y; George, M; Liu, J Q; Abbott, R J

2005-10-01

The vegetation of the northeast Qinghai-Tibetan Plateau is dominated by alpine meadow and desert-steppe with sparse forests scattered within it. To obtain a better understanding of the phylogeography of one constituent species of the forests in this region, we examined chloroplast trnT-trnF and trnS-trnG sequence variation within Juniperus przewalskii, a key endemic tree species. Sequence data were obtained from 392 trees in 20 populations covering the entire distribution range of the species. Six cpDNA haplotypes were identified. Significant population subdivision was detected (G(ST) = 0.772, N(ST) = 0.834), suggesting low levels of recurrent gene flow among populations and significant phylogeographic structure (N(ST) > G(ST), P < 0.05). Eight of the nine disjunct populations surveyed on the high-elevation northeast plateau were fixed for a single haplotype (A), while the remaining, more westerly population, contained the same haplotype at high frequency together with two low frequency haplotypes (C and F). In contrast, most populations that occurred at lower altitudes at the plateau edge were fixed or nearly fixed for one of two haplotypes, A or E. However, two plateau edge populations had haplotype compositions different from the rest. In one, four haplotypes (A, B, D and E) were present at approximately equivalent frequencies, which might reflect a larger refugium in the area of this population during the last glacial period. Phylogenetic analysis indicated that the most widely distributed haplotype A is not ancestral to other haplotypes. The contrasting phylogeographic structures of the haplotype-rich plateau edge area and the almost haplotype-uniform plateau platform region indicate that the plateau platform was recolonized by J. przewalskii during the most recent postglacial period. This is supported by the findings of a nested clade analysis, which inferred that postglacial range expansion from the plateau edge followed by recent fragmentation is largely responsible for the present-day spatial distribution of cpDNA haplotypes within the species.

Nanosilver-based surface-enhanced Raman spectroscopic determination of DNA methyltransferase activity through real-time hybridization chain reaction.

PubMed

Hu, Ping Ping; Liu, Hui; Zhen, Shu Jun; Li, Chun Mei; Huang, Cheng Zhi

2015-11-15

In this manuscript, a nanosilver enhanced SERS strategy was successfully constructed for the determination of DNA methyltransferase activity in soulution combined with hybridization chain reaction (HCR). The proposed method was mainly on the basis of excellent separation ability of magnetic microparticles (MMPs), HCR as signal amplification unit and assembled AgNPs as enhancement substrate. In the presence of M. SssI MTase, the duplex sequence (5'-CCGG-3') tethered to MMPs was methylated, which cannot be cleaved by HpaII endonuclease. The resulted DNA skeleton captured on MMPs then triggered the HCR reaction, generated a polymerized and extended symmetrical sequence, in which more biotin terminal was available for the conjugation of AgNPs-SA, leading to significantly amplified SERS response. When it was used to analyze M. SssI activity, a linear equation ∆ISERS=1215.32+446.80 cM.SssI was obtained with the M. SssI activity ranged from 0.1 to 10.0 U with the correlation coefficient (r(2)) of 0.97. The most important advantage of this method is the combination of SERS and HCR in solution for the first time and its good selectivity, which enabled the detection of even one-base mismatched sequence. The new assay method holds great promising application to be a versatile platform for sensitive, high-throughput detection, and the screening of new anticancer drugs on DNA MTase. Copyright © 2015 Elsevier B.V. All rights reserved.

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

PubMed Central

Martin, Jennifer A.; Smith, Joshua E.; Warren, Mercedes; Chávez, Jorge L.; Hagen, Joshua A.; Kelley-Loughnane, Nancy

2015-01-01

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids. PMID:25870978

Surface Modification of Silicon Pillar Arrays To Enhance Fluorescence Detection of Uranium and DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lincoln, Danielle R.; Charlton, Jennifer J.; Hatab, Nahla A.

There is an ever-growing need for detection methods that are both sensitive and efficient, such that reagent and sample consumption is minimized. Nanopillar arrays offer an attractive option to fill this need by virtue of their small scale in conjunction with their field enhancement intensity gains. This work investigates the use of nanopillar substrates for the detection of the uranyl ion and DNA, two analytes unalike but for their low quantum efficiencies combined with the need for high-throughput analyses. Here in this paper, the adaptability of these platforms was explored, as methods for the successful surface immobilization of both analytesmore » were developed and compared, resulting in a limit of detection for the uranyl ion of less than 1 ppm with a 0.2 μL sample volume. Moreover, differentiation between single-stranded and double-stranded DNA was possible, including qualitative identification between double-stranded DNA and DNA of the same sequence, but with a 10-base-pair mismatch.« less

Surface Modification of Silicon Pillar Arrays To Enhance Fluorescence Detection of Uranium and DNA

DOE PAGES

Lincoln, Danielle R.; Charlton, Jennifer J.; Hatab, Nahla A.; ...

2017-10-27

There is an ever-growing need for detection methods that are both sensitive and efficient, such that reagent and sample consumption is minimized. Nanopillar arrays offer an attractive option to fill this need by virtue of their small scale in conjunction with their field enhancement intensity gains. This work investigates the use of nanopillar substrates for the detection of the uranyl ion and DNA, two analytes unalike but for their low quantum efficiencies combined with the need for high-throughput analyses. Here in this paper, the adaptability of these platforms was explored, as methods for the successful surface immobilization of both analytesmore » were developed and compared, resulting in a limit of detection for the uranyl ion of less than 1 ppm with a 0.2 μL sample volume. Moreover, differentiation between single-stranded and double-stranded DNA was possible, including qualitative identification between double-stranded DNA and DNA of the same sequence, but with a 10-base-pair mismatch.« less

Launching genomics into the cloud: deployment of Mercury, a next generation sequence analysis pipeline

PubMed Central

2014-01-01

Background Massively parallel DNA sequencing generates staggering amounts of data. Decreasing cost, increasing throughput, and improved annotation have expanded the diversity of genomics applications in research and clinical practice. This expanding scale creates analytical challenges: accommodating peak compute demand, coordinating secure access for multiple analysts, and sharing validated tools and results. Results To address these challenges, we have developed the Mercury analysis pipeline and deployed it in local hardware and the Amazon Web Services cloud via the DNAnexus platform. Mercury is an automated, flexible, and extensible analysis workflow that provides accurate and reproducible genomic results at scales ranging from individuals to large cohorts. Conclusions By taking advantage of cloud computing and with Mercury implemented on the DNAnexus platform, we have demonstrated a powerful combination of a robust and fully validated software pipeline and a scalable computational resource that, to date, we have applied to more than 10,000 whole genome and whole exome samples. PMID:24475911

Endozoicomonas dominates the gill and intestinal content microbiomes of Mytilus edulis from Barnegat Bay, New Jersey

USGS Publications Warehouse

Schill, William B.; Iwanowicz, Deborah; Adams, Cynthia

2017-01-01

Blue mussels, Mytilus edulis, Linnaeus 1758 from southern Barnegat Bay, New Jersey were examined to determine the make-up of the normal blue mussel microbiome. Sequencing of 16S ribosomal DNA amplicons from gill and intestinal content microbiomes using the Illumina® MiSeq platform yielded 1,276,161 paired end sequence reads from the gill libraries and 1,092,333 paired end sequence reads from the intestinal content libraries. General bioinformatic analyses were conducted with the open-source packages Qiime and Mothur. Phylotype assignments to the genus level were made using the commercial One Codex platform. This resulted in 1,697,852 gill and 988,436 intestinal content sequences being classified to genus. A majority of these (67.6% and 37.2% respectively) were assigned to a single operational taxonomic unit (Mytilus edulis Symbiont, MeS) that has homologies with other recently described Endozoicomonas pathogens and symbionts of marine invertebrates. MeS shares 98% identity with an uncultured bacterium from the gill tissue of an invasive indo-Pacific oyster and with HQE1 and HQE2 isolated from the sea squirt, Styela clava. Other than MeS, most of the detected bacterial species are known from marine sediments and seawater.

Draft Genome of the Pearl Oyster Pinctada fucata: A Platform for Understanding Bivalve Biology

PubMed Central

Takeuchi, Takeshi; Kawashima, Takeshi; Koyanagi, Ryo; Gyoja, Fuki; Tanaka, Makiko; Ikuta, Tetsuro; Shoguchi, Eiichi; Fujiwara, Mayuki; Shinzato, Chuya; Hisata, Kanako; Fujie, Manabu; Usami, Takeshi; Nagai, Kiyohito; Maeyama, Kaoru; Okamoto, Kikuhiko; Aoki, Hideo; Ishikawa, Takashi; Masaoka, Tetsuji; Fujiwara, Atushi; Endo, Kazuyoshi; Endo, Hirotoshi; Nagasawa, Hiromichi; Kinoshita, Shigeharu; Asakawa, Shuichi; Watabe, Shugo; Satoh, Nori

2012-01-01

The study of the pearl oyster Pinctada fucata is key to increasing our understanding of the molecular mechanisms involved in pearl biosynthesis and biology of bivalve molluscs. We sequenced ∼1150-Mb genome at ∼40-fold coverage using the Roche 454 GS-FLX and Illumina GAIIx sequencers. The sequences were assembled into contigs with N50 = 1.6 kb (total contig assembly reached to 1024 Mb) and scaffolds with N50 = 14.5 kb. The pearl oyster genome is AT-rich, with a GC content of 34%. DNA transposons, retrotransposons, and tandem repeat elements occupied 0.4, 1.5, and 7.9% of the genome, respectively (a total of 9.8%). Version 1.0 of the P. fucata draft genome contains 23 257 complete gene models, 70% of which are supported by the corresponding expressed sequence tags. The genes include those reported to have an association with bio-mineralization. Genes encoding transcription factors and signal transduction molecules are present in numbers comparable with genomes of other metazoans. Genome-wide molecular phylogeny suggests that the lophotrochozoan represents a distinct clade from ecdysozoans. Our draft genome of the pearl oyster thus provides a platform for the identification of selection markers and genes for calcification, knowledge of which will be important in the pearl industry. PMID:22315334

Comparison of next generation sequencing technologies for transcriptome characterization

PubMed Central

2009-01-01

Background We have developed a simulation approach to help determine the optimal mixture of sequencing methods for most complete and cost effective transcriptome sequencing. We compared simulation results for traditional capillary sequencing with "Next Generation" (NG) ultra high-throughput technologies. The simulation model was parameterized using mappings of 130,000 cDNA sequence reads to the Arabidopsis genome (NCBI Accession SRA008180.19). We also generated 454-GS20 sequences and de novo assemblies for the basal eudicot California poppy (Eschscholzia californica) and the magnoliid avocado (Persea americana) using a variety of methods for cDNA synthesis. Results The Arabidopsis reads tagged more than 15,000 genes, including new splice variants and extended UTR regions. Of the total 134,791 reads (13.8 MB), 119,518 (88.7%) mapped exactly to known exons, while 1,117 (0.8%) mapped to introns, 11,524 (8.6%) spanned annotated intron/exon boundaries, and 3,066 (2.3%) extended beyond the end of annotated UTRs. Sequence-based inference of relative gene expression levels correlated significantly with microarray data. As expected, NG sequencing of normalized libraries tagged more genes than non-normalized libraries, although non-normalized libraries yielded more full-length cDNA sequences. The Arabidopsis data were used to simulate additional rounds of NG and traditional EST sequencing, and various combinations of each. Our simulations suggest a combination of FLX and Solexa sequencing for optimal transcriptome coverage at modest cost. We have also developed ESTcalc http://fgp.huck.psu.edu/NG_Sims/ngsim.pl, an online webtool, which allows users to explore the results of this study by specifying individualized costs and sequencing characteristics. Conclusion NG sequencing technologies are a highly flexible set of platforms that can be scaled to suit different project goals. In terms of sequence coverage alone, the NG sequencing is a dramatic advance over capillary-based sequencing, but NG sequencing also presents significant challenges in assembly and sequence accuracy due to short read lengths, method-specific sequencing errors, and the absence of physical clones. These problems may be overcome by hybrid sequencing strategies using a mixture of sequencing methodologies, by new assemblers, and by sequencing more deeply. Sequencing and microarray outcomes from multiple experiments suggest that our simulator will be useful for guiding NG transcriptome sequencing projects in a wide range of organisms. PMID:19646272

Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy).

PubMed

Garcia, Jessica; Forestier, Julien; Dusserre, Eric; Wozny, Anne-Sophie; Geiguer, Florence; Merle, Patrick; Tissot, Claire; Ferraro-Peyret, Carole; Jones, Frederick S; Edelstein, Daniel L; Cheynet, Valérie; Bardel, Claire; Vilchez, Gaelle; Xu, Zhenyu; Bringuier, Pierre Paul; Barritault, Marc; Brengle-Pesce, Karen; Guillet, Marielle; Chauvenet, Marion; Manship, Brigitte; Brevet, Marie; Rodriguez-Lafrasse, Claire; Hervieu, Valérie; Couraud, Sébastien; Walter, Thomas; Payen, Léa

2018-04-20

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5-1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%. Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results.

Cross-platform comparison for the detection of RAS mutations in cfDNA (ddPCR Biorad detection assay, BEAMing assay, and NGS strategy)

PubMed Central

Garcia, Jessica; Forestier, Julien; Dusserre, Eric; Wozny, Anne-Sophie; Geiguer, Florence; Merle, Patrick; Tissot, Claire; Ferraro-Peyret, Carole; Jones, Frederick S.; Edelstein, Daniel L.; Cheynet, Valérie; Bardel, Claire; Vilchez, Gaelle; Xu, Zhenyu; Bringuier, Pierre Paul; Barritault, Marc; Brengle-Pesce, Karen; Guillet, Marielle; Chauvenet, Marion; Manship, Brigitte; Brevet, Marie; Rodriguez-Lafrasse, Claire; Hervieu, Valérie; Couraud, Sébastien; Walter, Thomas; Payen, Léa

2018-01-01

CfDNA samples from colon (mCRC) and non-small cell lung cancers (NSCLC) (CIRCAN cohort) were compared using three platforms: droplet digital PCR (ddPCR, Biorad); BEAMing/OncoBEAM™-RAS-CRC (Sysmex Inostics); next-generation sequencing (NGS, Illumina), utilizing the 56G oncology panel (Swift Biosciences). Tissue biopsy and time matched cfDNA samples were collected at diagnosis in the mCRC cohort and during 1st progression in the NSCLC cohort. Excellent matches between cfDNA/FFPE mutation profiles were observed. Detection thresholds were between 0.5–1% for cfDNA samples examined using ddPCR and NGS, and 0.03% with BEAMing. This high level of sensitivity enabled the detection of KRAS mutations in 5/19 CRC patients with negative FFPE profiles. In the mCRC cohort, comparison of mutation results obtained by testing FFPE to those obtained by testing cfDNA by ddPCR resulted in 47% sensitivity, 77% specificity, 70% positive predictive value (PPV) and 55% negative predictive value (NPV). For BEAMing, we observed 93% sensitivity, 69% specificity, 78% PPV and 90% NPV. Finally, sensitivity of NGS was 73%, specificity was 77%, PPV 79% and NPV 71%. Our study highlights the complementarity of different diagnostic approaches and variability of results between OncoBEAM™-RAS-CRC and NGS assays. While the NGS assay provided a larger breadth of coverage of the major targetable alterations of 56 genes in one run, its performance for specific alterations was frequently confirmed by ddPCR results. PMID:29765524

Development of Single-Molecule DNA Sequencing Platform Based on Single-Molecule Electrical Conductance

DTIC Science & Technology

2015-05-25

nanoparticles , Nature Nanotechnology 7, 197-203. 11. Dreaden, E. C., Alkilany, A. M., Huang, X. H., Murphy, C. J., and El-Sayed, M. A. (2012) The...13840-13851. 14. Llevot, A., and Astruc, D. (2012) Applications of vectorized gold nanoparticles to the diagnosis and therapy of cancer , Chem. Soc. Rev...caused by the injection of gold nanoparticles , Nanotechnology 21, 485102. 25. Dykman, L. A., Matora, L. Y., and Bogatyrev, V. A. (1996) Use of

A Distributed Amplifier System for Bilayer Lipid Membrane (BLM) Arrays With Noise and Individual Offset Cancellation.

PubMed

Crescentini, Marco; Thei, Frederico; Bennati, Marco; Saha, Shimul; de Planque, Maurits R R; Morgan, Hywel; Tartagni, Marco

2015-06-01

Lipid bilayer membrane (BLM) arrays are required for high throughput analysis, for example drug screening or advanced DNA sequencing. Complex microfluidic devices are being developed but these are restricted in terms of array size and structure or have integrated electronic sensing with limited noise performance. We present a compact and scalable multichannel electrophysiology platform based on a hybrid approach that combines integrated state-of-the-art microelectronics with low-cost disposable fluidics providing a platform for high-quality parallel single ion channel recording. Specifically, we have developed a new integrated circuit amplifier based on a novel noise cancellation scheme that eliminates flicker noise derived from devices under test and amplifiers. The system is demonstrated through the simultaneous recording of ion channel activity from eight bilayer membranes. The platform is scalable and could be extended to much larger array sizes, limited only by electronic data decimation and communication capabilities.

A genome-wide BAC-end sequence survey provides first insights into sweetpotato (Ipomoea batatas (L.) Lam.) genome composition.

PubMed

Si, Zengzhi; Du, Bing; Huo, Jinxi; He, Shaozhen; Liu, Qingchang; Zhai, Hong

2016-11-21

Sweetpotato, Ipomoea batatas (L.) Lam., is an important food crop widely grown in the world. However, little is known about the genome of this species because it is a highly heterozygous hexaploid. Gaining a more in-depth knowledge of sweetpotato genome is therefore necessary and imperative. In this study, the first bacterial artificial chromosome (BAC) library of sweetpotato was constructed. Clones from the BAC library were end-sequenced and analyzed to provide genome-wide information about this species. The BAC library contained 240,384 clones with an average insert size of 101 kb and had a 7.93-10.82 × coverage of the genome, and the probability of isolating any single-copy DNA sequence from the library was more than 99%. Both ends of 8310 BAC clones randomly selected from the library were sequenced to generate 11,542 high-quality BAC-end sequences (BESs), with an accumulative length of 7,595,261 bp and an average length of 658 bp. Analysis of the BESs revealed that 12.17% of the sweetpotato genome were known repetitive DNA, including 7.37% long terminal repeat (LTR) retrotransposons, 1.15% Non-LTR retrotransposons and 1.42% Class II DNA transposons etc., 18.31% of the genome were identified as sweetpotato-unique repetitive DNA and 10.00% of the genome were predicted to be coding regions. In total, 3,846 simple sequences repeats (SSRs) were identified, with a density of one SSR per 1.93 kb, from which 288 SSRs primers were designed and tested for length polymorphism using 20 sweetpotato accessions, 173 (60.07%) of them produced polymorphic bands. Sweetpotato BESs had significant hits to the genome sequences of I. trifida and more matches to the whole-genome sequences of Solanum lycopersicum than those of Vitis vinifera, Theobroma cacao and Arabidopsis thaliana. The first BAC library for sweetpotato has been successfully constructed. The high quality BESs provide first insights into sweetpotato genome composition, and have significant hits to the genome sequences of I. trifida and more matches to the whole-genome sequences of Solanum lycopersicum. These resources as a robust platform will be used in high-resolution mapping, gene cloning, assembly of genome sequences, comparative genomics and evolution for sweetpotato.

Biocompatible nanostructured magnesium oxide-chitosan platform for genosensing application.

PubMed

Patel, Manoj Kumar; Ali, Md Azahar; Zafaryab, Md; Agrawal, Ved Varun; Rizvi, M Moshahid Alam; Ansari, Z A; Ansari, S G; Malhotra, Bansi D

2013-07-15

A novel organic-inorganic platform comprising of chitosan (CH) modified nanostructured magnesium oxide (nanoMgO) has been electrophoretically deposited on the indium-tin-oxide (ITO) substrate. The single stranded probe DNA (ssDNA) sequence of Vibrio cholerae has been covalently functionalized onto CH-nanoMgO/ITO surface. The cytotoxicity assay of nanoMgO particles, examined using human intestinal cell line (INT 407), reveals no significant cytotoxicity at the given doses in the range of 50-350 μg/mL. The X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR) and various microscopic techniques have been employed for the structural and morphological analysis of the fabricated electrodes. The electrochemical response studies of ssDNA and fragmented genomic DNA hybridized electrode (dsGDNA/CH-nanoMgO/ITO) have been carried out using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques. The dsGDNA/CH-nanoMgO/ITO bioelectrode exhibits a linear response in the range 100-500 ng/μL with improved sensitivity of 36.72 nA/ng/cm(2), faster response time of 3s and high stability of 3-4 months under refrigerated condition. The lower detection limit of fabricated genosensor has been estimated as 35.20 ng/μL and it shows good reproducibility/repeatability. Copyright © 2013 Elsevier B.V. All rights reserved.

Applying pollen DNA metabarcoding to the study of plant–pollinator interactions1

PubMed Central

Bell, Karen L.; Fowler, Julie; Burgess, Kevin S.; Dobbs, Emily K.; Gruenewald, David; Lawley, Brice; Morozumi, Connor; Brosi, Berry J.

2017-01-01

Premise of the study: To study pollination networks in a changing environment, we need accurate, high-throughput methods. Previous studies have shown that more highly resolved networks can be constructed by studying pollen loads taken from bees, relative to field observations. DNA metabarcoding potentially allows for faster and finer-scale taxonomic resolution of pollen compared to traditional approaches (e.g., light microscopy), but has not been applied to pollination networks. Methods: We sampled pollen from 38 bee species collected in Florida from sites differing in forest management. We isolated DNA from pollen mixtures and sequenced rbcL and ITS2 gene regions from all mixtures in a single run on the Illumina MiSeq platform. We identified species from sequence data using comprehensive rbcL and ITS2 databases. Results: We successfully built a proof-of-concept quantitative pollination network using pollen metabarcoding. Discussion: Our work underscores that pollen metabarcoding is not quantitative but that quantitative networks can be constructed based on the number of interacting individuals. Due to the frequency of contamination and false positive reads, isolation and PCR negative controls should be used in every reaction. DNA metabarcoding has advantages in efficiency and resolution over microscopic identification of pollen, and we expect that it will have broad utility for future studies of plant–pollinator interactions. PMID:28690929

Slowing DNA Translocation in a Nanofluidic Field-Effect Transistor.

PubMed

Liu, Yifan; Yobas, Levent

2016-04-26

Here, we present an experimental demonstration of slowing DNA translocation across a nanochannel by modulating the channel surface charge through an externally applied gate bias. The experiments were performed on a nanofluidic field-effect transistor, which is a monolithic integrated platform featuring a 50 nm-diameter in-plane alumina nanocapillary whose entire length is surrounded by a gate electrode. The field-effect transistor behavior was validated on the gating of ionic conductance and protein transport. The gating of DNA translocation was subsequently studied by measuring discrete current dips associated with single λ-DNA translocation events under a source-to-drain bias of 1 V. The translocation speeds under various gate bias conditions were extracted by fitting event histograms of the measured translocation time to the first passage time distributions obtained from a simple 1D biased diffusion model. A positive gate bias was observed to slow the translocation of single λ-DNA chains markedly; the translocation speed was reduced by an order of magnitude from 18.4 mm/s obtained under a floating gate down to 1.33 mm/s under a positive gate bias of 9 V. Therefore, a dynamic and flexible regulation of the DNA translocation speed, which is vital for single-molecule sequencing, can be achieved on this device by simply tuning the gate bias. The device is realized in a conventional semiconductor microfabrication process without the requirement of advanced lithography, and can be potentially further developed into a compact electronic single-molecule sequencer.

Bacteriophage T4 capsid packaging and unpackaging of DNA and proteins.

PubMed

Mullaney, Julienne M; Black, Lindsay W

2014-01-01

Bacteriophage T4 has proven itself readily amenable to phage-based DNA and protein packaging, expression, and display systems due to its physical resiliency and genomic flexibility. As a large dsDNA phage with dispensable internal proteins and dispensable outer capsid proteins it can be adapted to package both DNA and proteins of interest within the capsid and to display peptides and proteins externally on the capsid. A single 170 kb linear DNA, or single or multiple copies of shorter linear DNAs, of any sequence can be packaged by the large terminase subunit in vitro into protein-containing proheads and give full or partially full capsids. The prohead receptacles for DNA packaging can also display peptides or full-length proteins from capsid display proteins HOC and SOC. Our laboratory has also developed a protein expression, packaging, and processing (PEPP) system which we have found to have advantages over mammalian and bacterial cell systems, including high yield, increased stability, and simplified downstream processing. Proteins that we have produced by the phage PEPP platform include human HIV-1 protease, micrococcal endonuclease from Staphylococcus aureus, restriction endonuclease EcoRI, luciferase, human granulocyte colony stimulating factor (GCSF), green fluorescent protein (GFP), and the 99 amino acid C-terminus of amyloid precursor protein (APP). Difficult to produce proteins that are toxic in mammalian protein expression systems are easily produced, packaged, and processed with the PEPP platform. APP is one example of such a highly refractory protein that has been produced successfully. The methods below describe the procedures for in vitro packaging of proheads with DNA and for producing recombinant T4 phage that carry a gene of interest in the phage genome and produce and internally package the corresponding protein of interest.

Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine-needle aspirate samples.

PubMed

Dyhdalo, Kathryn; Macnamara, Stephen; Brainard, Jennifer; Underwood, Dawn; Tubbs, Raymond; Yang, Bin

2014-02-01

BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society. © 2013 American Cancer Society.

Assessment of the cPAS-based BGISEQ-500 platform for metagenomic sequencing.

PubMed

Fang, Chao; Zhong, Huanzi; Lin, Yuxiang; Chen, Bing; Han, Mo; Ren, Huahui; Lu, Haorong; Luber, Jacob M; Xia, Min; Li, Wangsheng; Stein, Shayna; Xu, Xun; Zhang, Wenwei; Drmanac, Radoje; Wang, Jian; Yang, Huanming; Hammarström, Lennart; Kostic, Aleksandar D; Kristiansen, Karsten; Li, Junhua

2018-03-01

More extensive use of metagenomic shotgun sequencing in microbiome research relies on the development of high-throughput, cost-effective sequencing. Here we present a comprehensive evaluation of the performance of the new high-throughput sequencing platform BGISEQ-500 for metagenomic shotgun sequencing and compare its performance with that of 2 Illumina platforms. Using fecal samples from 20 healthy individuals, we evaluated the intra-platform reproducibility for metagenomic sequencing on the BGISEQ-500 platform in a setup comprising 8 library replicates and 8 sequencing replicates. Cross-platform consistency was evaluated by comparing 20 pairwise replicates on the BGISEQ-500 platform vs the Illumina HiSeq 2000 platform and the Illumina HiSeq 4000 platform. In addition, we compared the performance of the 2 Illumina platforms against each other. By a newly developed overall accuracy quality control method, an average of 82.45 million high-quality reads (96.06% of raw reads) per sample, with 90.56% of bases scoring Q30 and above, was obtained using the BGISEQ-500 platform. Quantitative analyses revealed extremely high reproducibility between BGISEQ-500 intra-platform replicates. Cross-platform replicates differed slightly more than intra-platform replicates, yet a high consistency was observed. Only a low percentage (2.02%-3.25%) of genes exhibited significant differences in relative abundance comparing the BGISEQ-500 and HiSeq platforms, with a bias toward genes with higher GC content being enriched on the HiSeq platforms. Our study provides the first set of performance metrics for human gut metagenomic sequencing data using BGISEQ-500. The high accuracy and technical reproducibility confirm the applicability of the new platform for metagenomic studies, though caution is still warranted when combining metagenomic data from different platforms.

Genovo: De Novo Assembly for Metagenomes

NASA Astrophysics Data System (ADS)

Laserson, Jonathan; Jojic, Vladimir; Koller, Daphne

Next-generation sequencing technologies produce a large number of noisy reads from the DNA in a sample. Metagenomics and population sequencing aim to recover the genomic sequences of the species in the sample, which could be of high diversity. Methods geared towards single sequence reconstruction are not sensitive enough when applied in this setting. We introduce a generative probabilistic model of read generation from environmental samples and present Genovo, a novel de novo sequence assembler that discovers likely sequence reconstructions under the model. A Chinese restaurant process prior accounts for the unknown number of genomes in the sample. Inference is made by applying a series of hill-climbing steps iteratively until convergence. We compare the performance of Genovo to three other short read assembly programs across one synthetic dataset and eight metagenomic datasets created using the 454 platform, the largest of which has 311k reads. Genovo's reconstructions cover more bases and recover more genes than the other methods, and yield a higher assembly score.

Next-generation sequencing: the future of molecular genetics in poultry production and food safety.

PubMed

Diaz-Sanchez, S; Hanning, I; Pendleton, Sean; D'Souza, Doris

2013-02-01

The era of molecular biology and automation of the Sanger chain-terminator sequencing method has led to discovery and advances in diagnostics and biotechnology. The Sanger methodology dominated research for over 2 decades, leading to significant accomplishments and technological improvements in DNA sequencing. Next-generation high-throughput sequencing (HT-NGS) technologies were developed subsequently to overcome the limitations of this first generation technology that include higher speed, less labor, and lowered cost. Various platforms developed include sequencing-by-synthesis 454 Life Sciences, Illumina (Solexa) sequencing, SOLiD sequencing (among others), and the Ion Torrent semiconductor sequencing technologies that use different detection principles. As technology advances, progress made toward third generation sequencing technologies are being reported, which include Nanopore Sequencing and real-time monitoring of PCR activity through fluorescent resonant energy transfer. The advantages of these technologies include scalability, simplicity, with increasing DNA polymerase performance and yields, being less error prone, and even more economically feasible with the eventual goal of obtaining real-time results. These technologies can be directly applied to improve poultry production and enhance food safety. For example, sequence-based (determination of the gut microbial community, genes for metabolic pathways, or presence of plasmids) and function-based (screening for function such as antibiotic resistance, or vitamin production) metagenomic analysis can be carried out. Gut microbialflora/communities of poultry can be sequenced to determine the changes that affect health and disease along with efficacy of methods to control pathogenic growth. Thus, the purpose of this review is to provide an overview of the principles of these current technologies and their potential application to improve poultry production and food safety as well as public health.

Quantitation of heteroplasmy of mtDNA sequence variants identified in a population of AD patients and controls by array-based resequencing.

PubMed

Coon, Keith D; Valla, Jon; Szelinger, Szabolics; Schneider, Lonnie E; Niedzielko, Tracy L; Brown, Kevin M; Pearson, John V; Halperin, Rebecca; Dunckley, Travis; Papassotiropoulos, Andreas; Caselli, Richard J; Reiman, Eric M; Stephan, Dietrich A

2006-08-01

The role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease (AD) has been well documented. Though evidence for the role of mitochondria in AD seems incontrovertible, the impact of mitochondrial DNA (mtDNA) mutations in AD etiology remains controversial. Though mutations in mitochondrially encoded genes have repeatedly been implicated in the pathogenesis of AD, many of these studies have been plagued by lack of replication as well as potential contamination of nuclear-encoded mitochondrial pseudogenes. To assess the role of mtDNA mutations in the pathogenesis of AD, while avoiding the pitfalls of nuclear-encoded mitochondrial pseudogenes encountered in previous investigations and showcasing the benefits of a novel resequencing technology, we sequenced the entire coding region (15,452 bp) of mtDNA from 19 extremely well-characterized AD patients and 18 age-matched, unaffected controls utilizing a new, reliable, high-throughput array-based resequencing technique, the Human MitoChip. High-throughput, array-based DNA resequencing of the entire mtDNA coding region from platelets of 37 subjects revealed the presence of 208 loci displaying a total of 917 sequence variants. There were no statistically significant differences in overall mutational burden between cases and controls, however, 265 independent sites of statistically significant change between cases and controls were identified. Changed sites were found in genes associated with complexes I (30.2%), III (3.0%), IV (33.2%), and V (9.1%) as well as tRNA (10.6%) and rRNA (14.0%). Despite their statistical significance, the subtle nature of the observed changes makes it difficult to determine whether they represent true functional variants involved in AD etiology or merely naturally occurring dissimilarity. Regardless, this study demonstrates the tremendous value of this novel mtDNA resequencing platform, which avoids the pitfalls of erroneously amplifying nuclear-encoded mtDNA pseudogenes, and our proposed analysis paradigm, which utilizes the availability of raw signal intensity values for each of the four potential alleles to facilitate quantitative estimates of mtDNA heteroplasmy. This information provides a potential new target for burgeoning diagnostics and therapeutics that could truly assist those suffering from this devastating disorder.

Services of DNA barcoding in different fields.

PubMed

Muhammad Tahir, Hafiz; Akhtar, Samreen

2016-11-01

DNA barcoding is a new master key for species identification and has greatly accelerated the pace of species discovery. In this novel and cost-effective technique, a short DNA sequence from a standard region of mitochondrial "CO1" gene called "barcode" is used. At present, researchers all over the world are utilizing this powerful tool for investigating biodiversity, differentiating cryptic species, testing food authenticity, identifying parasites, vectors, insect pests, and predators, monitoring of illegal trade of animals and their products, and identifying forensically important insects. In addition, this technique can potentially be used to monitor quality of drinking water, quickly identify the indicator species of lakes, rivers, and streams, identify species with harmful attributes or medicinal properties, monitor smuggling of endangered plants and animals and their products, and disease investigations. Despite non-favorable criticism from a few researchers, DNA barcoding has achieved immense popularity in the scientific community, especially among biologists. The present review provides an overview of DNA barcoding and its practical applications. The limitation, future prospective and main informative platforms for DNA barcoding have also been discussed.

DNA biosensors implemented on PNA-functionalized microstructured optical fibers Bragg gratings

NASA Astrophysics Data System (ADS)

Candiani, A.; Giannetti, S.; Cucinotta, A.; Bertucci, A.; Manicardi, A.; Konstantaki, M.; Margulis, W.; Pissadakis, S.; Corradini, R.; Selleri, S.

2013-05-01

A novel DNA sensing platform based on a Peptide Nucleic Acid - functionalized Microstructured Optical Fibers gratings has been demonstrated. The inner surface of different MOFs has been functionalized using PNA probes, OligoNucleotides mimic that are well suited for specific DNA target sequences detection. The hybrid sensing systems were tested for optical DNA detection of targets of relevance in biomedical application, using the cystic fibrosis gene mutation, and food-analysis, using the genomic DNA from genetic modified organism soy flour. After the solutions of DNA molecules has been infiltrated inside the fibers capillaries and hybridization has occurred, oligonucleotidefunctionalized gold nanoparticles were infiltrated and used to form a sandwich-like system to achieve signal amplification. Spectral measurements of the reflected signal reveal a clear wavelength shift of the reflected modes when the infiltrated complementary DNA matches with the PNA probes placed on the inner fiber surface. Measurements have also been made using the mismatched DNA solution for the c, containing a single nucleotide polymorphism, showing no significant changes in the reflected spectrum. Several experiments have been carried out demonstrating the reproducibility of the results and the high selectivity of the sensors, showing the simplicity and the potential of this approach.

Genomic resources for Myzus persicae: EST sequencing, SNP identification, and microarray design

PubMed Central

Ramsey, John S; Wilson, Alex CC; de Vos, Martin; Sun, Qi; Tamborindeguy, Cecilia; Winfield, Agnese; Malloch, Gaynor; Smith, Dawn M; Fenton, Brian; Gray, Stewart M; Jander, Georg

2007-01-01

Background The green peach aphid, Myzus persicae (Sulzer), is a world-wide insect pest capable of infesting more than 40 plant families, including many crop species. However, despite the significant damage inflicted by M. persicae in agricultural systems through direct feeding damage and by its ability to transmit plant viruses, limited genomic information is available for this species. Results Sequencing of 16 M. persicae cDNA libraries generated 26,669 expressed sequence tags (ESTs). Aphids for library construction were raised on Arabidopsis thaliana, Nicotiana benthamiana, Brassica oleracea, B. napus, and Physalis floridana (with and without Potato leafroll virus infection). The M. persicae cDNA libraries include ones made from sexual and asexual whole aphids, guts, heads, and salivary glands. In silico comparison of cDNA libraries identified aphid genes with tissue-specific expression patterns, and gene expression that is induced by feeding on Nicotiana benthamiana. Furthermore, 2423 genes that are novel to science and potentially aphid-specific were identified. Comparison of cDNA data from three aphid lineages identified single nucleotide polymorphisms that can be used as genetic markers and, in some cases, may represent functional differences in the protein products. In particular, non-conservative amino acid substitutions in a highly expressed gut protease may be of adaptive significance for M. persicae feeding on different host plants. The Agilent eArray platform was used to design an M. persicae oligonucleotide microarray representing over 10,000 unique genes. Conclusion New genomic resources have been developed for M. persicae, an agriculturally important insect pest. These include previously unknown sequence data, a collection of expressed genes, molecular markers, and a DNA microarray that can be used to study aphid gene expression. These resources will help elucidate the adaptations that allow M. persicae to develop compatible interactions with its host plants, complementing ongoing work illuminating plant molecular responses to phloem-feeding insects. PMID:18021414

PR-PR: Cross-Platform Laboratory Automation System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Linshiz, G; Stawski, N; Goyal, G

To enable protocol standardization, sharing, and efficient implementation across laboratory automation platforms, we have further developed the PR-PR open-source high-level biology-friendly robot programming language as a cross-platform laboratory automation system. Beyond liquid-handling robotics, PR-PR now supports microfluidic and microscopy platforms, as well as protocol translation into human languages, such as English. While the same set of basic PR-PR commands and features are available for each supported platform, the underlying optimization and translation modules vary from platform to platform. Here, we describe these further developments to PR-PR, and demonstrate the experimental implementation and validation of PR-PR protocols for combinatorial modified Goldenmore » Gate DNA assembly across liquid-handling robotic, microfluidic, and manual platforms. To further test PR-PR cross-platform performance, we then implement and assess PR-PR protocols for Kunkel DNA mutagenesis and hierarchical Gibson DNA assembly for microfluidic and manual platforms.« less

PR-PR: cross-platform laboratory automation system.

PubMed

Linshiz, Gregory; Stawski, Nina; Goyal, Garima; Bi, Changhao; Poust, Sean; Sharma, Monica; Mutalik, Vivek; Keasling, Jay D; Hillson, Nathan J

2014-08-15

To enable protocol standardization, sharing, and efficient implementation across laboratory automation platforms, we have further developed the PR-PR open-source high-level biology-friendly robot programming language as a cross-platform laboratory automation system. Beyond liquid-handling robotics, PR-PR now supports microfluidic and microscopy platforms, as well as protocol translation into human languages, such as English. While the same set of basic PR-PR commands and features are available for each supported platform, the underlying optimization and translation modules vary from platform to platform. Here, we describe these further developments to PR-PR, and demonstrate the experimental implementation and validation of PR-PR protocols for combinatorial modified Golden Gate DNA assembly across liquid-handling robotic, microfluidic, and manual platforms. To further test PR-PR cross-platform performance, we then implement and assess PR-PR protocols for Kunkel DNA mutagenesis and hierarchical Gibson DNA assembly for microfluidic and manual platforms.

Open-target sparse sensing of biological agents using DNA microarray

PubMed Central

2011-01-01

Background Current biosensors are designed to target and react to specific nucleic acid sequences or structural epitopes. These 'target-specific' platforms require creation of new physical capture reagents when new organisms are targeted. An 'open-target' approach to DNA microarray biosensing is proposed and substantiated using laboratory generated data. The microarray consisted of 12,900 25 bp oligonucleotide capture probes derived from a statistical model trained on randomly selected genomic segments of pathogenic prokaryotic organisms. Open-target detection of organisms was accomplished using a reference library of hybridization patterns for three test organisms whose DNA sequences were not included in the design of the microarray probes. Results A multivariate mathematical model based on the partial least squares regression (PLSR) was developed to detect the presence of three test organisms in mixed samples. When all 12,900 probes were used, the model correctly detected the signature of three test organisms in all mixed samples (mean(R2)) = 0.76, CI = 0.95), with a 6% false positive rate. A sampling algorithm was then developed to sparsely sample the probe space for a minimal number of probes required to capture the hybridization imprints of the test organisms. The PLSR detection model was capable of correctly identifying the presence of the three test organisms in all mixed samples using only 47 probes (mean(R2)) = 0.77, CI = 0.95) with nearly 100% specificity. Conclusions We conceived an 'open-target' approach to biosensing, and hypothesized that a relatively small, non-specifically designed, DNA microarray is capable of identifying the presence of multiple organisms in mixed samples. Coupled with a mathematical model applied to laboratory generated data, and sparse sampling of capture probes, the prototype microarray platform was able to capture the signature of each organism in all mixed samples with high sensitivity and specificity. It was demonstrated that this new approach to biosensing closely follows the principles of sparse sensing. PMID:21801424

Integrated massively parallel sequencing of 15 autosomal STRs and Amelogenin using a simplified library preparation approach.

PubMed

Xue, Jian; Wu, Riga; Pan, Yajiao; Wang, Shunxia; Qu, Baowang; Qin, Ying; Shi, Yuequn; Zhang, Chuchu; Li, Ran; Zhang, Liyan; Zhou, Cheng; Sun, Hongyu

2018-04-02

Massively parallel sequencing (MPS) technologies, also termed as next-generation sequencing (NGS), are becoming increasingly popular in study of short tandem repeats (STR). However, current library preparation methods are usually based on ligation or two-round PCR that requires more steps, making it time-consuming (about 2 days), laborious and expensive. In this study, a 16-plex STR typing system was designed with fusion primer strategy based on the Ion Torrent S5 XL platform which could effectively resolve the above challenges for forensic DNA database-type samples (bloodstains, saliva stains, etc.). The efficiency of this system was tested in 253 Han Chinese participants. The libraries were prepared without DNA isolation and adapter ligation, and the whole process only required approximately 5 h. The proportion of thoroughly genotyped samples in which all the 16 loci were successfully genotyped was 86% (220/256). Of the samples, 99.7% showed 100% concordance between NGS-based STR typing and capillary electrophoresis (CE)-based STR typing. The inconsistency might have been caused by off-ladder alleles and mutations in primer binding sites. Overall, this panel enabled the large-scale genotyping of the DNA samples with controlled quality and quantity because it is a simple, operation-friendly process flow that saves labor, time and costs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris

PubMed Central

Zhao, Yanan; Lockhart, Shawn R.; Berrio, Indira

2017-01-01

ABSTRACT Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii, Candida haemulonii, and Candida lusitaniae. Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species. PMID:28539346

Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris.

PubMed

Kordalewska, Milena; Zhao, Yanan; Lockhart, Shawn R; Chowdhary, Anuradha; Berrio, Indira; Perlin, David S

2017-08-01

Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii , Candida haemulonii , and Candida lusitaniae Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species. Copyright © 2017 Kordalewska et al.

The Ancient Evolutionary History of Polyomaviruses

PubMed Central

Buck, Christopher B.; Van Doorslaer, Koenraad; Peretti, Alberto; Geoghegan, Eileen M.; Tisza, Michael J.; An, Ping; Katz, Joshua P.; Pipas, James M.; McBride, Alison A.; Camus, Alvin C.; McDermott, Alexa J.; Dill, Jennifer A.; Delwart, Eric; Ng, Terry F. F.; Farkas, Kata; Austin, Charlotte; Kraberger, Simona; Davison, William; Pastrana, Diana V.; Varsani, Arvind

2016-01-01

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae. PMID:27093155

gkmSVM: an R package for gapped-kmer SVM

PubMed Central

Ghandi, Mahmoud; Mohammad-Noori, Morteza; Ghareghani, Narges; Lee, Dongwon; Garraway, Levi; Beer, Michael A.

2016-01-01

Summary: We present a new R package for training gapped-kmer SVM classifiers for DNA and protein sequences. We describe an improved algorithm for kernel matrix calculation that speeds run time by about 2 to 5-fold over our original gkmSVM algorithm. This package supports several sequence kernels, including: gkmSVM, kmer-SVM, mismatch kernel and wildcard kernel. Availability and Implementation: gkmSVM package is freely available through the Comprehensive R Archive Network (CRAN), for Linux, Mac OS and Windows platforms. The C ++ implementation is available at www.beerlab.org/gkmsvm Contact: mghandi@gmail.com or mbeer@jhu.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153639

An innovative SNP genotyping method adapting to multiple platforms and throughputs.

PubMed

Long, Y M; Chao, W S; Ma, G J; Xu, S S; Qi, L L

2017-03-01

An innovative genotyping method designated as semi-thermal asymmetric reverse PCR (STARP) was developed for genotyping individual SNPs with improved accuracy, flexible throughputs, low operational costs, and high platform compatibility. Multiplex chip-based technology for genome-scale genotyping of single nucleotide polymorphisms (SNPs) has made great progress in the past two decades. However, PCR-based genotyping of individual SNPs still remains problematic in accuracy, throughput, simplicity, and/or operational costs as well as the compatibility with multiple platforms. Here, we report a novel SNP genotyping method designated semi-thermal asymmetric reverse PCR (STARP). In this method, genotyping assay was performed under unique PCR conditions using two universal priming element-adjustable primers (PEA-primers) and one group of three locus-specific primers: two asymmetrically modified allele-specific primers (AMAS-primers) and their common reverse primer. The two AMAS-primers each were substituted one base in different positions at their 3' regions to significantly increase the amplification specificity of the two alleles and tailed at 5' ends to provide priming sites for PEA-primers. The two PEA-primers were developed for common use in all genotyping assays to stringently target the PCR fragments generated by the two AMAS-primers with similar PCR efficiencies and for flexible detection using either gel-free fluorescence signals or gel-based size separation. The state-of-the-art primer design and unique PCR conditions endowed STARP with all the major advantages of high accuracy, flexible throughputs, simple assay design, low operational costs, and platform compatibility. In addition to SNPs, STARP can also be employed in genotyping of indels (insertion-deletion polymorphisms). As vast variations in DNA sequences are being unearthed by many genome sequencing projects and genotyping by sequencing, STARP will have wide applications across all biological organisms in agriculture, medicine, and forensics.

Phylogenetic analysis of higher-level relationships within Hydroidolina (Cnidaria: Hydrozoa) using mitochondrial genome data and insight into their mitochondrial transcription.

PubMed

Kayal, Ehsan; Bentlage, Bastian; Cartwright, Paulyn; Yanagihara, Angel A; Lindsay, Dhugal J; Hopcroft, Russell R; Collins, Allen G

2015-01-01

Hydrozoans display the most morphological diversity within the phylum Cnidaria. While recent molecular studies have provided some insights into their evolutionary history, sister group relationships remain mostly unresolved, particularly at mid-taxonomic levels. Specifically, within Hydroidolina, the most speciose hydrozoan subclass, the relationships and sometimes integrity of orders are highly unsettled. Here we obtained the near complete mitochondrial sequence of twenty-six hydroidolinan hydrozoan species from a range of sources (DNA and RNA-seq data, long-range PCR). Our analyses confirm previous inference of the evolution of mtDNA in Hydrozoa while introducing a novel genome organization. Using RNA-seq data, we propose a mechanism for the expression of mitochondrial mRNA in Hydroidolina that can be extrapolated to the other medusozoan taxa. Phylogenetic analyses using the full set of mitochondrial gene sequences provide some insights into the order-level relationships within Hydroidolina, including siphonophores as the first diverging clade, a well-supported clade comprised of Leptothecata-Filifera III-IV, and a second clade comprised of Aplanulata-Capitata s.s.-Filifera I-II. Finally, we describe our relatively inexpensive and accessible multiplexing strategy to sequence long-range PCR amplicons that can be adapted to most high-throughput sequencing platforms.

Phylogenetic analysis of higher-level relationships within Hydroidolina (Cnidaria: Hydrozoa) using mitochondrial genome data and insight into their mitochondrial transcription

PubMed Central

Bentlage, Bastian; Cartwright, Paulyn; Yanagihara, Angel A.; Lindsay, Dhugal J.; Hopcroft, Russell R.; Collins, Allen G.

2015-01-01

Hydrozoans display the most morphological diversity within the phylum Cnidaria. While recent molecular studies have provided some insights into their evolutionary history, sister group relationships remain mostly unresolved, particularly at mid-taxonomic levels. Specifically, within Hydroidolina, the most speciose hydrozoan subclass, the relationships and sometimes integrity of orders are highly unsettled. Here we obtained the near complete mitochondrial sequence of twenty-six hydroidolinan hydrozoan species from a range of sources (DNA and RNA-seq data, long-range PCR). Our analyses confirm previous inference of the evolution of mtDNA in Hydrozoa while introducing a novel genome organization. Using RNA-seq data, we propose a mechanism for the expression of mitochondrial mRNA in Hydroidolina that can be extrapolated to the other medusozoan taxa. Phylogenetic analyses using the full set of mitochondrial gene sequences provide some insights into the order-level relationships within Hydroidolina, including siphonophores as the first diverging clade, a well-supported clade comprised of Leptothecata-Filifera III–IV, and a second clade comprised of Aplanulata-Capitata s.s.-Filifera I–II. Finally, we describe our relatively inexpensive and accessible multiplexing strategy to sequence long-range PCR amplicons that can be adapted to most high-throughput sequencing platforms. PMID:26618080

Genome-Wide Stochastic Adaptive DNA Amplification at Direct and Inverted DNA Repeats in the Parasite Leishmania

PubMed Central

Plourde, Marie; Gingras, Hélène; Roy, Gaétan; Lapointe, Andréanne; Leprohon, Philippe; Papadopoulou, Barbara; Corbeil, Jacques; Ouellette, Marc

2014-01-01

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment. PMID:24844805

Identification and characterization of Highlands J virus from a Mississippi sandhill crane using unbiased next-generation sequencing

USGS Publications Warehouse

Ip, Hon S.; Wiley, Michael R.; Long, Renee; Gustavo, Palacios; Shearn-Bochsler, Valerie; Whitehouse, Chris A.

2014-01-01

Advances in massively parallel DNA sequencing platforms, commonly termed next-generation sequencing (NGS) technologies, have greatly reduced time, labor, and cost associated with DNA sequencing. Thus, NGS has become a routine tool for new viral pathogen discovery and will likely become the standard for routine laboratory diagnostics of infectious diseases in the near future. This study demonstrated the application of NGS for the rapid identification and characterization of a virus isolated from the brain of an endangered Mississippi sandhill crane. This bird was part of a population restoration effort and was found in an emaciated state several days after Hurricane Isaac passed over the refuge in Mississippi in 2012. Post-mortem examination had identified trichostrongyliasis as the possible cause of death, but because a virus with morphology consistent with a togavirus was isolated from the brain of the bird, an arboviral etiology was strongly suspected. Because individual molecular assays for several known arboviruses were negative, unbiased NGS by Illumina MiSeq was used to definitively identify and characterize the causative viral agent. Whole genome sequencing and phylogenetic analysis revealed the viral isolate to be the Highlands J virus, a known avian pathogen. This study demonstrates the use of unbiased NGS for the rapid detection and characterization of an unidentified viral pathogen and the application of this technology to wildlife disease diagnostics and conservation medicine.

The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies.

PubMed

Yoshida, Catherine E; Kruczkiewicz, Peter; Laing, Chad R; Lingohr, Erika J; Gannon, Victor P J; Nash, John H E; Taboada, Eduardo N

2016-01-01

For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-genome sequencing (WGS) promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR), a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence-based methods of sub-typing allows for continuity with historical serotyping data as we transition towards the increasing adoption of genomic analyses in epidemiology. The SISTR platform is freely available on the web at https://lfz.corefacility.ca/sistr-app/.

ParticleCall: A particle filter for base calling in next-generation sequencing systems

PubMed Central

2012-01-01

Background Next-generation sequencing systems are capable of rapid and cost-effective DNA sequencing, thus enabling routine sequencing tasks and taking us one step closer to personalized medicine. Accuracy and lengths of their reads, however, are yet to surpass those provided by the conventional Sanger sequencing method. This motivates the search for computationally efficient algorithms capable of reliable and accurate detection of the order of nucleotides in short DNA fragments from the acquired data. Results In this paper, we consider Illumina’s sequencing-by-synthesis platform which relies on reversible terminator chemistry and describe the acquired signal by reformulating its mathematical model as a Hidden Markov Model. Relying on this model and sequential Monte Carlo methods, we develop a parameter estimation and base calling scheme called ParticleCall. ParticleCall is tested on a data set obtained by sequencing phiX174 bacteriophage using Illumina’s Genome Analyzer II. The results show that the developed base calling scheme is significantly more computationally efficient than the best performing unsupervised method currently available, while achieving the same accuracy. Conclusions The proposed ParticleCall provides more accurate calls than the Illumina’s base calling algorithm, Bustard. At the same time, ParticleCall is significantly more computationally efficient than other recent schemes with similar performance, rendering it more feasible for high-throughput sequencing data analysis. Improvement of base calling accuracy will have immediate beneficial effects on the performance of downstream applications such as SNP and genotype calling. ParticleCall is freely available at https://sourceforge.net/projects/particlecall. PMID:22776067

Detection of Bacillus anthracis DNA in Complex Soil and Air Samples Using Next-Generation Sequencing

PubMed Central

Be, Nicholas A.; Thissen, James B.; Gardner, Shea N.; McLoughlin, Kevin S.; Fofanov, Viacheslav Y.; Koshinsky, Heather; Ellingson, Sally R.; Brettin, Thomas S.; Jackson, Paul J.; Jaing, Crystal J.

2013-01-01

Bacillus anthracis is the potentially lethal etiologic agent of anthrax disease, and is a significant concern in the realm of biodefense. One of the cornerstones of an effective biodefense strategy is the ability to detect infectious agents with a high degree of sensitivity and specificity in the context of a complex sample background. The nature of the B. anthracis genome, however, renders specific detection difficult, due to close homology with B. cereus and B. thuringiensis. We therefore elected to determine the efficacy of next-generation sequencing analysis and microarrays for detection of B. anthracis in an environmental background. We applied next-generation sequencing to titrated genome copy numbers of B. anthracis in the presence of background nucleic acid extracted from aerosol and soil samples. We found next-generation sequencing to be capable of detecting as few as 10 genomic equivalents of B. anthracis DNA per nanogram of background nucleic acid. Detection was accomplished by mapping reads to either a defined subset of reference genomes or to the full GenBank database. Moreover, sequence data obtained from B. anthracis could be reliably distinguished from sequence data mapping to either B. cereus or B. thuringiensis. We also demonstrated the efficacy of a microbial census microarray in detecting B. anthracis in the same samples, representing a cost-effective and high-throughput approach, complementary to next-generation sequencing. Our results, in combination with the capacity of sequencing for providing insights into the genomic characteristics of complex and novel organisms, suggest that these platforms should be considered important components of a biosurveillance strategy. PMID:24039948

Coverage Bias and Sensitivity of Variant Calling for Four Whole-genome Sequencing Technologies

PubMed Central

Lasitschka, Bärbel; Jones, David; Northcott, Paul; Hutter, Barbara; Jäger, Natalie; Kool, Marcel; Taylor, Michael; Lichter, Peter; Pfister, Stefan; Wolf, Stephan; Brors, Benedikt; Eils, Roland

2013-01-01

The emergence of high-throughput, next-generation sequencing technologies has dramatically altered the way we assess genomes in population genetics and in cancer genomics. Currently, there are four commonly used whole-genome sequencing platforms on the market: Illumina’s HiSeq2000, Life Technologies’ SOLiD 4 and its completely redesigned 5500xl SOLiD, and Complete Genomics’ technology. A number of earlier studies have compared a subset of those sequencing platforms or compared those platforms with Sanger sequencing, which is prohibitively expensive for whole genome studies. Here we present a detailed comparison of the performance of all currently available whole genome sequencing platforms, especially regarding their ability to call SNVs and to evenly cover the genome and specific genomic regions. Unlike earlier studies, we base our comparison on four different samples, allowing us to assess the between-sample variation of the platforms. We find a pronounced GC bias in GC-rich regions for Life Technologies’ platforms, with Complete Genomics performing best here, while we see the least bias in GC-poor regions for HiSeq2000 and 5500xl. HiSeq2000 gives the most uniform coverage and displays the least sample-to-sample variation. In contrast, Complete Genomics exhibits by far the smallest fraction of bases not covered, while the SOLiD platforms reveal remarkable shortcomings, especially in covering CpG islands. When comparing the performance of the four platforms for calling SNPs, HiSeq2000 and Complete Genomics achieve the highest sensitivity, while the SOLiD platforms show the lowest false positive rate. Finally, we find that integrating sequencing data from different platforms offers the potential to combine the strengths of different technologies. In summary, our results detail the strengths and weaknesses of all four whole-genome sequencing platforms. It indicates application areas that call for a specific sequencing platform and disallow other platforms. This helps to identify the proper sequencing platform for whole genome studies with different application scopes. PMID:23776689

An ultrasensitive label-free biosensor for assaying of sequence-specific DNA-binding protein based on amplifying fluorescent conjugated polymer.

PubMed

Liu, Xingfen; Ouyang, Lan; Cai, Xiaohui; Huang, Yanqin; Feng, Xiaomiao; Fan, Quli; Huang, Wei

2013-03-15

Sensitive, reliable, and simple detection of sequence-specific DNA-binding proteins (DBP) is of paramount importance in the area of proteomics, genomics, and biomedicine. We describe herein a novel fluorescent-amplified strategy for ultrasensitive, visual, quantitative, and "turn-on" detection of DBP. A Förster resonance energy transfer (FRET) assay utilizing a cationic conjugated polymer (CCP) and an intercalating dye was designed to detect a key transcription factor, nuclear factor-kappa B (NF-κB), the model target. A series of label-free DNA probes bearing one or two protein-binding sites (PBS) were used to identify the target protein specifically. The binding DBP protects the probe from digestion by exonuclease III, resulting in high efficient FRET due to the high affinity between the intercalating dye and duplex DNA, as well as strong electrostatic interactions between the CCP and DNA probe. By using label-free hairpin DNA or double-stranded DNA containing two PBS as probe, we could detect as low as 1 pg/μL of NF-κB in HeLa nuclear extracts, which is 10000-fold more sensitive than the previously reported methods. The approach also allows naked-eye detection by observing fluorescent color of solutions with the assistance of a hand-held UV lamp. Additionally, a less than 10% relative standard deviation was obtained, which offers a new platform for superior precision, low-cost, and simple detection of DBP. The features of our optical biosensor shows promising potential for early diagnosis of many diseases and high-throughput screening of new drugs targeted to DNA-binding proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

High-throughput microsatellite genotyping in ecology: improved accuracy, efficiency, standardization and success with low-quantity and degraded DNA.

PubMed

De Barba, M; Miquel, C; Lobréaux, S; Quenette, P Y; Swenson, J E; Taberlet, P

2017-05-01

Microsatellite markers have played a major role in ecological, evolutionary and conservation research during the past 20 years. However, technical constrains related to the use of capillary electrophoresis and a recent technological revolution that has impacted other marker types have brought to question the continued use of microsatellites for certain applications. We present a study for improving microsatellite genotyping in ecology using high-throughput sequencing (HTS). This approach entails selection of short markers suitable for HTS, sequencing PCR-amplified microsatellites on an Illumina platform and bioinformatic treatment of the sequence data to obtain multilocus genotypes. It takes advantage of the fact that HTS gives direct access to microsatellite sequences, allowing unambiguous allele identification and enabling automation of the genotyping process through bioinformatics. In addition, the massive parallel sequencing abilities expand the information content of single experimental runs far beyond capillary electrophoresis. We illustrated the method by genotyping brown bear samples amplified with a multiplex PCR of 13 new microsatellite markers and a sex marker. HTS of microsatellites provided accurate individual identification and parentage assignment and resulted in a significant improvement of genotyping success (84%) of faecal degraded DNA and costs reduction compared to capillary electrophoresis. The HTS approach holds vast potential for improving success, accuracy, efficiency and standardization of microsatellite genotyping in ecological and conservation applications, especially those that rely on profiling of low-quantity/quality DNA and on the construction of genetic databases. We discuss and give perspectives for the implementation of the method in the light of the challenges encountered in wildlife studies. © 2016 John Wiley & Sons Ltd.

miR-ID: A novel, circularization-based platform for detection of microRNAs

PubMed Central

Kumar, Pavan; Johnston, Brian H.; Kazakov, Sergei A.

2011-01-01

MicroRNAs (miRNAs) are important regulators of gene expression and have great potential as biomarkers, prognostic indicators, and therapeutic targets. Determining the expression patterns of these molecules is essential for elucidating their biogenesis, regulation, relation to disease, and response to therapy. Although PCR-based assays are commonly used for expression profiling of miRNAs, the small size, sequence heterogeneity, and (in some cases) end modifications of miRNAs constrain the performance of existing PCR methods. Here we introduce miR-ID, a novel method that avoids these constraints while providing superior sensitivity and sequence specificity at a lower cost. It also has the unique ability to differentiate unmodified small RNAs from those carrying 2′-OMe groups at their 3′-ends while detecting both forms. miR-ID is comprised of the following steps: (1) circularization of the miRNA by a ligase; (2) reverse transcription of the circularized miRNA (RTC), producing tandem repeats of a DNA sequence complementary to the miRNA; and (3) qPCR amplification of segments of this multimeric cDNA using 5′-overlapping primers and a nonspecific dye such as SYBR Green. No chemically modified probes (e.g., TaqMan) or primers (e.g., LNA) are required. The circular RNA and multimeric cDNA templates provide unmatched flexibility in the positioning of primers, which may include straddling the boundaries between these repetitive miRNA sequences. miR-ID is based on new findings that are themselves of general interest, including reverse transcription of small RNA circles and the use of 5′-overlapping primers for detection of repetitive sequences by qPCR. PMID:21169480

A Single-Molecule Barcoding System using Nanoslits for DNA Analysis

NASA Astrophysics Data System (ADS)

Jo, Kyubong; Schramm, Timothy M.; Schwartz, David C.

Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels, creating molecular barcodes, which are efficiently read using fluorescence resonance energy transfer techniques for minimizing noise from unincorporated labels. As such, our integrative approach for the realization of genomic analysis through nanoconfinement, named nanocoding, was demonstrated through the barcoding and mapping of bacterial artificial chromosomal molecules, thereby providing the basis for a high-throughput platform competent for whole genome investigations.

RNA-seq Analysis Reveals Unique Transcriptome Signatures in Systemic Lupus Erythematosus Patients with Distinct Autoantibody Specificities

PubMed Central

Rai, Richa; Chauhan, Sudhir Kumar; Singh, Vikas Vikram; Rai, Madhukar; Rai, Geeta

2016-01-01

Systemic lupus erythematosus (SLE) patients exhibit immense heterogeneity which is challenging from the diagnostic perspective. Emerging high throughput sequencing technologies have been proved to be a useful platform to understand the complex and dynamic disease processes. SLE patients categorised based on autoantibody specificities are reported to have differential immuno-regulatory mechanisms. Therefore, we performed RNA-seq analysis to identify transcriptomics of SLE patients with distinguished autoantibody specificities. The SLE patients were segregated into three subsets based on the type of autoantibodies present in their sera (anti-dsDNA+ group with anti-dsDNA autoantibody alone; anti-ENA+ group having autoantibodies against extractable nuclear antigens (ENA) only, and anti-dsDNA+ENA+ group having autoantibodies to both dsDNA and ENA). Global transcriptome profiling for each SLE patients subsets was performed using Illumina® Hiseq-2000 platform. The biological relevance of dysregulated transcripts in each SLE subsets was assessed by ingenuity pathway analysis (IPA) software. We observed that dysregulation in the transcriptome expression pattern was clearly distinct in each SLE patients subsets. IPA analysis of transcripts uniquely expressed in different SLE groups revealed specific biological pathways to be affected in each SLE subsets. Multiple cytokine signaling pathways were specifically dysregulated in anti-dsDNA+ patients whereas Interferon signaling was predominantly dysregulated in anti-ENA+ patients. In anti-dsDNA+ENA+ patients regulation of actin based motility by Rho pathway was significantly affected. The granulocyte gene signature was a common feature to all SLE subsets; however, anti-dsDNA+ group showed relatively predominant expression of these genes. Dysregulation of Plasma cell related transcripts were higher in anti-dsDNA+ and anti-ENA+ patients as compared to anti-dsDNA+ ENA+. Association of specific canonical pathways with the uniquely expressed transcripts in each SLE subgroup indicates that specific immunological disease mechanisms are operative in distinct SLE patients’ subsets. This ‘sub-grouping’ approach could further be useful for clinical evaluation of SLE patients and devising targeted therapeutics. PMID:27835693

Target-triggering multiple-cycle signal amplification strategy for ultrasensitive detection of DNA based on QCM and SPR.

PubMed

Song, Weiling; Yin, Wenshuo; Sun, Wenbo; Guo, Xiaoyan; He, Peng; Yang, Xiaoyan; Zhang, Xiaoru

2018-04-24

Detection of ultralow concentrations of nucleic acid sequences is a central challenge in the early diagnosis of genetic diseases. Herein, we developed a target-triggering cascade multiple cycle amplification for ultrasensitive DNA detection using quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). It was based on the exonuclease Ⅲ (Exo Ⅲ)-assisted signal amplification and the hybridization chain reaction (HCR). The streptavidin-coated Au-NPs (Au-NPs-SA) were assembled on the HCR products as recognition element. Upon sensing of target DNA, the duplex DNA probe triggered the Exo Ⅲ cleavage process, accompanied by generating a new secondary target DNA and releasing target DNA. The released target DNA and the secondary target DNA were recycled. Simultaneously, numerous single strands were liberated and acted as the trigger of HCR to generate further signal amplification, resulting in the immobilization of abundant Au-NPs-SA on the gold substrate. The QCM sensor results were found to be comparable to that achieved using a SPR sensor platform. This method exhibited a high sensitivity toward target DNA with a detection limit of 0.70 fM. The high sensitivity and specificity make this method a great potential for detecting DNA with trace amounts in bioanalysis and clinical biomedicine. Copyright © 2018 Elsevier Inc. All rights reserved.

Current and Emerging Technologies for the Analysis of the Genome-Wide and Locus-Specific DNA Methylation Patterns.

PubMed

Tost, Jörg

2016-01-01

DNA methylation is the most studied epigenetic modification, and altered DNA methylation patterns have been identified in cancer and more recently also in many other complex diseases. Furthermore, DNA methylation is influenced by a variety of environmental factors, and the analysis of DNA methylation patterns might allow deciphering previous exposure. Although a large number of techniques to study DNA methylation either genome-wide or at specific loci have been devised, they all are based on a limited number of principles for differentiating the methylation state, viz., methylation-specific/methylation-dependent restriction enzymes, antibodies or methyl-binding proteins, chemical-based enrichment, or bisulfite conversion. Second-generation sequencing has largely replaced microarrays as readout platform and is also becoming more popular for locus-specific DNA methylation analysis. In this chapter, the currently used methods for both genome-wide and locus-specific analysis of 5-methylcytosine and as its oxidative derivatives, such as 5-hydroxymethylcytosine, are reviewed in detail, and the advantages and limitations of each approach are discussed. Furthermore, emerging technologies avoiding PCR amplification and allowing a direct readout of DNA methylation are summarized, together with novel applications, such as the detection of DNA methylation in single cells or in circulating cell-free DNA.

Engineering M13 for phage display.

PubMed

Sidhu, S S

2001-09-01

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.

An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

PubMed Central

Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

2011-01-01

Background Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. Methodology/Principal Findings We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Conclusions/Significance Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories. PMID:21637859

Designing deep sequencing experiments: detecting structural variation and estimating transcript abundance.

PubMed

Bashir, Ali; Bansal, Vikas; Bafna, Vineet

2010-06-18

Massively parallel DNA sequencing technologies have enabled the sequencing of several individual human genomes. These technologies are also being used in novel ways for mRNA expression profiling, genome-wide discovery of transcription-factor binding sites, small RNA discovery, etc. The multitude of sequencing platforms, each with their unique characteristics, pose a number of design challenges, regarding the technology to be used and the depth of sequencing required for a particular sequencing application. Here we describe a number of analytical and empirical results to address design questions for two applications: detection of structural variations from paired-end sequencing and estimating mRNA transcript abundance. For structural variation, our results provide explicit trade-offs between the detection and resolution of rearrangement breakpoints, and the optimal mix of paired-read insert lengths. Specifically, we prove that optimal detection and resolution of breakpoints is achieved using a mix of exactly two insert library lengths. Furthermore, we derive explicit formulae to determine these insert length combinations, enabling a 15% improvement in breakpoint detection at the same experimental cost. On empirical short read data, these predictions show good concordance with Illumina 200 bp and 2 Kbp insert length libraries. For transcriptome sequencing, we determine the sequencing depth needed to detect rare transcripts from a small pilot study. With only 1 Million reads, we derive corrections that enable almost perfect prediction of the underlying expression probability distribution, and use this to predict the sequencing depth required to detect low expressed genes with greater than 95% probability. Together, our results form a generic framework for many design considerations related to high-throughput sequencing. We provide software tools http://bix.ucsd.edu/projects/NGS-DesignTools to derive platform independent guidelines for designing sequencing experiments (amount of sequencing, choice of insert length, mix of libraries) for novel applications of next generation sequencing.

Molecular Mapping of Restriction-Site Associated DNA Markers In Allotetraploid Upland Cotton.

PubMed

Wang, Yangkun; Ning, Zhiyuan; Hu, Yan; Chen, Jiedan; Zhao, Rui; Chen, Hong; Ai, Nijiang; Guo, Wangzhen; Zhang, Tianzhen

2015-01-01

Upland cotton (Gossypium hirsutum L., 2n = 52, AADD) is an allotetraploid, therefore the discovery of single nucleotide polymorphism (SNP) markers is difficult. The recent emergence of genome complexity reduction technologies based on the next-generation sequencing (NGS) platform has greatly expedited SNP discovery in crops with highly repetitive and complex genomes. Here we applied restriction-site associated DNA (RAD) sequencing technology for de novo SNP discovery in allotetraploid cotton. We identified 21,109 SNPs between the two parents and used these for genotyping of 161 recombinant inbred lines (RILs). Finally, a high dense linkage map comprising 4,153 loci over 3500-cM was developed based on the previous result. Using this map quantitative trait locus (QTLs) conferring fiber strength and Verticillium Wilt (VW) resistance were mapped to a more accurate region in comparison to the 1576-cM interval determined using the simple sequence repeat (SSR) genetic map. This suggests that the newly constructed map has more power and resolution than the previous SSR map. It will pave the way for the rapid identification of the marker-assisted selection in cotton breeding and cloning of QTL of interest traits.

Myopathology of Adult and Paediatric Mitochondrial Diseases

PubMed Central

Phadke, Rahul

2017-01-01

Mitochondria are dynamic organelles ubiquitously present in nucleated eukaryotic cells, subserving multiple metabolic functions, including cellular ATP generation by oxidative phosphorylation (OXPHOS). The OXPHOS machinery comprises five transmembrane respiratory chain enzyme complexes (RC). Defective OXPHOS gives rise to mitochondrial diseases (mtD). The incredible phenotypic and genetic diversity of mtD can be attributed at least in part to the RC dual genetic control (nuclear DNA (nDNA) and mitochondrial DNA (mtDNA)) and the complex interaction between the two genomes. Despite the increasing use of next-generation-sequencing (NGS) and various omics platforms in unravelling novel mtD genes and pathomechanisms, current clinical practice for investigating mtD essentially involves a multipronged approach including clinical assessment, metabolic screening, imaging, pathological, biochemical and functional testing to guide molecular genetic analysis. This review addresses the broad muscle pathology landscape including genotype–phenotype correlations in adult and paediatric mtD, the role of immunodiagnostics in understanding some of the pathomechanisms underpinning the canonical features of mtD, and recent diagnostic advances in the field. PMID:28677615

Structural Insights into the Quadruplex-Duplex 3' Interface Formed from a Telomeric Repeat: A Potential Molecular Target.

PubMed

Russo Krauss, Irene; Ramaswamy, Sneha; Neidle, Stephen; Haider, Shozeb; Parkinson, Gary N

2016-02-03

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

Disentangling the many layers of eukaryotic transcriptional regulation.

PubMed

Lelli, Katherine M; Slattery, Matthew; Mann, Richard S

2012-01-01

Regulation of gene expression in eukaryotes is an extremely complex process. In this review, we break down several critical steps, emphasizing new data and techniques that have expanded current gene regulatory models. We begin at the level of DNA sequence where cis-regulatory modules (CRMs) provide important regulatory information in the form of transcription factor (TF) binding sites. In this respect, CRMs function as instructional platforms for the assembly of gene regulatory complexes. We discuss multiple mechanisms controlling complex assembly, including cooperative DNA binding, combinatorial codes, and CRM architecture. The second section of this review places CRM assembly in the context of nucleosomes and condensed chromatin. We discuss how DNA accessibility and histone modifications contribute to TF function. Lastly, new advances in chromosomal mapping techniques have provided increased understanding of intra- and interchromosomal interactions. We discuss how these topological maps influence gene regulatory models.

Assessment of replicate bias in 454 pyrosequencing and a multi-purpose read-filtering tool.

PubMed

Jérôme, Mariette; Noirot, Céline; Klopp, Christophe

2011-05-26

Roche 454 pyrosequencing platform is often considered the most versatile of the Next Generation Sequencing technology platforms, permitting the sequencing of large genomes, the analysis of variations or the study of transcriptomes. A recent reported bias leads to the production of multiple reads for a unique DNA fragment in a random manner within a run. This bias has a direct impact on the quality of the measurement of the representation of the fragments using the reads. Other cleaning steps are usually performed on the reads before assembly or alignment. PyroCleaner is a software module intended to clean 454 pyrosequencing reads in order to ease the assembly process. This program is a free software and is distributed under the terms of the GNU General Public License as published by the Free Software Foundation. It implements several filters using criteria such as read duplication, length, complexity, base-pair quality and number of undetermined bases. It also permits to clean flowgram files (.sff) of paired-end sequences generating on one hand validated paired-ends file and the other hand single read file. Read cleaning has always been an important step in sequence analysis. The pyrocleaner python module is a Swiss knife dedicated to 454 reads cleaning. It includes commonly used filters as well as specialised ones such as duplicated read removal and paired-end read verification.

HEx: A heterologous expression platform for the discovery of fungal natural products

PubMed Central

Schlecht, Ulrich; Horecka, Joe; Lin, Hsiao-Ching; Naughton, Brian; Miranda, Molly; Li, Yong Fuga; Hennessy, James R.; Vandova, Gergana A.; Steinmetz, Lars M.; Sattely, Elizabeth; Khosla, Chaitan; Hillenmeyer, Maureen E.

2018-01-01

For decades, fungi have been a source of U.S. Food and Drug Administration–approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products. PMID:29651464

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

PubMed Central

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-01-01

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013

A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets.

PubMed

Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

2014-12-05

Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways.

Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA.

PubMed

Haque, Farzin; Li, Jinghong; Wu, Hai-Chen; Liang, Xing-Jie; Guo, Peixuan

2013-02-01

Sensitivity and specificity are two most important factors to take into account for molecule sensing, chemical detection and disease diagnosis. A perfect sensitivity is to reach the level where a single molecule can be detected. An ideal specificity is to reach the level where the substance can be detected in the presence of many contaminants. The rapidly progressing nanopore technology is approaching this threshold. A wide assortment of biomotors and cellular pores in living organisms perform diverse biological functions. The elegant design of these transportation machineries has inspired the development of single molecule detection based on modulations of the individual current blockage events. The dynamic growth of nanotechnology and nanobiotechnology has stimulated rapid advances in the study of nanopore based instrumentation over the last decade, and inspired great interest in sensing of single molecules including ions, nucleotides, enantiomers, drugs, and polymers such as PEG, RNA, DNA, and polypeptides. This sensing technology has been extended to medical diagnostics and third generation high throughput DNA sequencing. This review covers current nanopore detection platforms including both biological pores and solid state counterparts. Several biological nanopores have been studied over the years, but this review will focus on the three best characterized systems including α-hemolysin and MspA, both containing a smaller channel for the detection of single-strand DNA, as well as bacteriophage phi29 DNA packaging motor connector that contains a larger channel for the passing of double stranded DNA. The advantage and disadvantage of each system are compared; their current and potential applications in nanomedicine, biotechnology, and nanotechnology are discussed.

Solid-State and Biological Nanopore for Real-Time Sensing of Single Chemical and Sequencing of DNA

PubMed Central

Haque, Farzin; Li, Jinghong; Wu, Hai-Chen; Liang, Xing-Jie; Guo, Peixuan

2013-01-01

Sensitivity and specificity are two most important factors to take into account for molecule sensing, chemical detection and disease diagnosis. A perfect sensitivity is to reach the level where a single molecule can be detected. An ideal specificity is to reach the level where the substance can be detected in the presence of many contaminants. The rapidly progressing nanopore technology is approaching this threshold. A wide assortment of biomotors and cellular pores in living organisms perform diverse biological functions. The elegant design of these transportation machineries has inspired the development of single molecule detection based on modulations of the individual current blockage events. The dynamic growth of nanotechnology and nanobiotechnology has stimulated rapid advances in the study of nanopore based instrumentation over the last decade, and inspired great interest in sensing of single molecules including ions, nucleotides, enantiomers, drugs, and polymers such as PEG, RNA, DNA, and polypeptides. This sensing technology has been extended to medical diagnostics and third generation high throughput DNA sequencing. This review covers current nanopore detection platforms including both biological pores and solid state counterparts. Several biological nanopores have been studied over the years, but this review will focus on the three best characterized systems including α-hemolysin and MspA, both containing a smaller channel for the detection of single-strand DNA, as well as bacteriophage phi29 DNA packaging motor connector that contains a larger channel for the passing of double stranded DNA. The advantage and disadvantage of each system are compared; their current and potential applications in nanomedicine, biotechnology, and nanotechnology are discussed. PMID:23504223

Molecular beacons for DNA binding proteins: an emerging technology for detection of DNA binding proteins and their ligands.

PubMed

Dummitt, Benjamin; Chang, Yie-Hwa

2006-06-01

Quantitation of the level or activity of specific proteins is one of the most commonly performed experiments in biomedical research. Protein detection has historically been difficult to adapt to high throughput platforms because of heavy reliance upon antibodies for protein detection. Molecular beacons for DNA binding proteins is a recently developed technology that attempts to overcome such limitations. Protein detection is accomplished using inexpensive, easy-to-synthesize oligonucleotides, accompanied by a fluorescence readout. Importantly, detection of the protein and reporting of the signal occur simultaneously, allowing for one-step protocols and increased potential for use in high throughput analysis. While the initial iteration of the technology allowed only for the detection of sequence-specific DNA binding proteins, more recent adaptations allow for the possibility of development of beacons for any protein, independent of native DNA binding activity. Here, we discuss the development of the technology, the mechanism of the reaction, and recent improvements and modifications made to improve the assay in terms of sensitivity, potential for multiplexing, and broad applicability.

Optimizing Illumina next-generation sequencing library preparation for extremely AT-biased genomes.

PubMed

Oyola, Samuel O; Otto, Thomas D; Gu, Yong; Maslen, Gareth; Manske, Magnus; Campino, Susana; Turner, Daniel J; Macinnis, Bronwyn; Kwiatkowski, Dominic P; Swerdlow, Harold P; Quail, Michael A

2012-01-03

Massively parallel sequencing technology is revolutionizing approaches to genomic and genetic research. Since its advent, the scale and efficiency of Next-Generation Sequencing (NGS) has rapidly improved. In spite of this success, sequencing genomes or genomic regions with extremely biased base composition is still a great challenge to the currently available NGS platforms. The genomes of some important pathogenic organisms like Plasmodium falciparum (high AT content) and Mycobacterium tuberculosis (high GC content) display extremes of base composition. The standard library preparation procedures that employ PCR amplification have been shown to cause uneven read coverage particularly across AT and GC rich regions, leading to problems in genome assembly and variation analyses. Alternative library-preparation approaches that omit PCR amplification require large quantities of starting material and hence are not suitable for small amounts of DNA/RNA such as those from clinical isolates. We have developed and optimized library-preparation procedures suitable for low quantity starting material and tolerant to extremely high AT content sequences. We have used our optimized conditions in parallel with standard methods to prepare Illumina sequencing libraries from a non-clinical and a clinical isolate (containing ~53% host contamination). By analyzing and comparing the quality of sequence data generated, we show that our optimized conditions that involve a PCR additive (TMAC), produces amplified libraries with improved coverage of extremely AT-rich regions and reduced bias toward GC neutral templates. We have developed a robust and optimized Next-Generation Sequencing library amplification method suitable for extremely AT-rich genomes. The new amplification conditions significantly reduce bias and retain the complexity of either extremes of base composition. This development will greatly benefit sequencing clinical samples that often require amplification due to low mass of DNA starting material.

Whole genome sequence phylogenetic analysis of four Mexican rabies viruses isolated from cattle.

PubMed

Bárcenas-Reyes, I; Loza-Rubio, E; Cantó-Alarcón, G J; Luna-Cozar, J; Enríquez-Vázquez, A; Barrón-Rodríguez, R J; Milián-Suazo, F

2017-08-01

Phylogenetic analysis of the rabies virus in molecular epidemiology has been traditionally performed on partial sequences of the genome, such as the N, G, and P genes; however, that approach raises concerns about the discriminatory power compared to whole genome sequencing. In this study we characterized four strains of the rabies virus isolated from cattle in Querétaro, Mexico by comparing the whole genome sequence to that of strains from the American, European and Asian continents. Four cattle brain samples positive to rabies and characterized as AgV11, genotype 1, were used in the study. A cDNA sequence was generated by reverse transcription PCR (RT-PCR) using oligo dT. cDNA samples were sequenced in an Illumina NextSeq 500 platform. The phylogenetic analysis was performed with MEGA 6.0. Minimum evolution phylogenetic trees were constructed with the Neighbor-Joining method and bootstrapped with 1000 replicates. Three large and seven small clusters were formed with the 26 sequences used. The largest cluster grouped strains from different species in South America: Brazil, and the French Guyana. The second cluster grouped five strains from Mexico. A Mexican strain reported in a different study was highly related to our four strains, suggesting common source of infection. The phylogenetic analysis shows that the type of host is different for the different regions in the American Continent; rabies is more related to bats. It was concluded that the rabies virus in central Mexico is genetically stable and that it is transmitted by the vampire bat Desmodus rotundus. Copyright © 2017 Elsevier Ltd. All rights reserved.

Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

PubMed Central

Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

2010-01-01

In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers. PMID:20941376

Sequence-specific sepsis-related DNA capture and fluorescent labeling in monoliths prepared by single-step photopolymerization in microfluidic devices.

PubMed

Knob, Radim; Hanson, Robert L; Tateoka, Olivia B; Wood, Ryan L; Guerrero-Arguero, Israel; Robison, Richard A; Pitt, William G; Woolley, Adam T

2018-05-21

Fast determination of antibiotic resistance is crucial in selecting appropriate treatment for sepsis patients, but current methods based on culture are time consuming. We are developing a microfluidic platform with a monolithic column modified with oligonucleotides designed for sequence-specific capture of target DNA related to the Klebsiella pneumoniae carbapenemase (KPC) gene. We developed a novel single-step monolith fabrication method with an acrydite-modified capture oligonucleotide in the polymerization mixture, enabling fast monolith preparation in a microfluidic channel using UV photopolymerization. These prepared columns had a threefold higher capacity compared to monoliths prepared in a multistep process involving Schiff-base DNA attachment. Conditions for denaturing, capture and fluorescence labeling using hybridization probes were optimized with synthetic 90-mer oligonucleotides. These procedures were applied for extraction of a PCR amplicon from the KPC antibiotic resistance gene in bacterial lysate obtained from a blood sample spiked with E. coli. The results showed similar eluted peak areas for KPC amplicon extracted from either hybridization buffer or bacterial lysate. Selective extraction of the KPC DNA was verified by real time PCR on eluted fractions. These results show great promise for application in an integrated microfluidic diagnostic system that combines upstream blood sample preparation and downstream single-molecule counting detection. Copyright © 2018 Elsevier B.V. All rights reserved.

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples

PubMed Central

Nguyen, David; Valenzuela, Nicole; Takemura, Ping; Bolon, Yung-Tsi; Springer, Brianna; Saito, Katsuyuki; Zheng, Ying; Hague, Tim; Pasztor, Agnes; Horvath, Gyorgy; Rigo, Krisztina; Reed, Elaine F.; Zhang, Qiuheng

2016-01-01

Background Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. Methods Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3’ UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. Results Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). Conclusion Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation. PMID:27798706

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples.

PubMed

Yin, Yuxin; Lan, James H; Nguyen, David; Valenzuela, Nicole; Takemura, Ping; Bolon, Yung-Tsi; Springer, Brianna; Saito, Katsuyuki; Zheng, Ying; Hague, Tim; Pasztor, Agnes; Horvath, Gyorgy; Rigo, Krisztina; Reed, Elaine F; Zhang, Qiuheng

2016-01-01

Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3' UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.

Single-cell barcoding and sequencing using droplet microfluidics.

PubMed

Zilionis, Rapolas; Nainys, Juozas; Veres, Adrian; Savova, Virginia; Zemmour, David; Klein, Allon M; Mazutis, Linas

2017-01-01

Single-cell RNA sequencing has recently emerged as a powerful tool for mapping cellular heterogeneity in diseased and healthy tissues, yet high-throughput methods are needed for capturing the unbiased diversity of cells. Droplet microfluidics is among the most promising candidates for capturing and processing thousands of individual cells for whole-transcriptome or genomic analysis in a massively parallel manner with minimal reagent use. We recently established a method called inDrops, which has the capability to index >15,000 cells in an hour. A suspension of cells is first encapsulated into nanoliter droplets with hydrogel beads (HBs) bearing barcoding DNA primers. Cells are then lysed and mRNA is barcoded (indexed) by a reverse transcription (RT) reaction. Here we provide details for (i) establishing an inDrops platform (1 d); (ii) performing hydrogel bead synthesis (4 d); (iii) encapsulating and barcoding cells (1 d); and (iv) RNA-seq library preparation (2 d). inDrops is a robust and scalable platform, and it is unique in its ability to capture and profile >75% of cells in even very small samples, on a scale of thousands or tens of thousands of cells.

Massively Parallel Sequencing of Forensic STRs Using the Ion Chef™ and the Ion S5™ XL Systems.

PubMed

Wang, Le; Chen, Man; Wu, Bo; Liu, Yi-Cheng; Zhang, Guang-Feng; Jiang, Li; Xu, Xiu-Lan; Zhao, Xing-Chun; Ji, An-Quan; Ye, Jian

2018-03-01

Next-generation sequencing (NGS) has been used to genotype forensic short tandem repeat (STR) markers for individual identification and kinship analysis. STR data from several NGS platforms have been published, but forensic application trials using the Ion S5™ XL system have not been reported. In this work, we report sensitivity, reproducibility, mixture, simulated degradation, and casework sample data on the Ion Chef™ and S5™ XL systems using an early access 25-plex panel. Sensitivity experiments showed that over 97% of the alleles were detectable with down to 62 pg input of genomic DNA. In mixture studies, alleles from minor contributors were correctly assigned at 1:9 and 9:1 ratios. NGS successfully gave 12 full genotype results from 13 challenging casework samples, compared with five full results using the CE platform. In conclusion, the Ion Chef™ and the Ion S5™ XL systems provided an alternative and promising approach for forensic STR genotyping. © 2018 American Academy of Forensic Sciences.

DNA barcoding the native flowering plants and conifers of Wales.

PubMed

de Vere, Natasha; Rich, Tim C G; Ford, Col R; Trinder, Sarah A; Long, Charlotte; Moore, Chris W; Satterthwaite, Danielle; Davies, Helena; Allainguillaume, Joel; Ronca, Sandra; Tatarinova, Tatiana; Garbett, Hannah; Walker, Kevin; Wilkinson, Mike J

2012-01-01

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification.

A Versatile Nanowire Platform for Highly Efficient Isolation and Direct PCR-free Colorimetric Detection of Human Papillomavirus DNA from Unprocessed Urine

PubMed Central

Lee, HyungJae; Choi, Mihye; Hwang, Sang-Hyun; Cho, Youngnam

2018-01-01

Purpose: As human papillomavirus (HPV) is primarily responsible for the development of cervical cancer, significant efforts have been devoted to develop novel strategies for detecting and identifying HPV DNA in urine. The analysis of target DNA sequences in urine offers a potential alternative to conventional methods as a non-invasive clinical screening and diagnostic assessment tool for the detection of HPV. However, the lack of efficient approaches to isolate and directly detect HPV DNA in urine has restricted its potential clinical use. In this study, we demonstrated a novel approach of using polyethylenimine-conjugated magnetic polypyrrole nanowires (PEI-mPpy NWs) for the extraction, identification, and PCR-free colorimetric detection of high-risk strains of HPV DNA sequences, particularly HPV-16 and HPV-18, in urine specimens of cervical cancer patients. Materials and Methods: We fabricated and characterized polyethylenimine-conjugated magnetic nanowires (PEI/mPpy NWs). PEI/mPpy NWs-based HPV DNA isolation and detection strategy appears to be a cost-effective and practical technology with greater sensitivity and accuracy than other urine-based methods. Results: The analytical and clinical performance of PEI-mPpy NWs was evaluated and compared with those of cervical swabs, demonstrating a superior type-specific concordance rate of 100% between urine and cervical swabs, even when using a small volume of urine (300 µL). Conclusion: We envision that PEI-mPpy NWs provide substantive evidence for clinical diagnosis and management of HPV-associated disease with their excellent performance in the recovery and detection of HPV DNA from minimal amounts of urine samples. PMID:29290816

DNA Barcoding the Native Flowering Plants and Conifers of Wales

PubMed Central

de Vere, Natasha; Rich, Tim C. G.; Ford, Col R.; Trinder, Sarah A.; Long, Charlotte; Moore, Chris W.; Satterthwaite, Danielle; Davies, Helena; Allainguillaume, Joel; Ronca, Sandra; Tatarinova, Tatiana; Garbett, Hannah; Walker, Kevin; Wilkinson, Mike J.

2012-01-01

We present the first national DNA barcode resource that covers the native flowering plants and conifers for the nation of Wales (1143 species). Using the plant DNA barcode markers rbcL and matK, we have assembled 97.7% coverage for rbcL, 90.2% for matK, and a dual-locus barcode for 89.7% of the native Welsh flora. We have sampled multiple individuals for each species, resulting in 3304 rbcL and 2419 matK sequences. The majority of our samples (85%) are from DNA extracted from herbarium specimens. Recoverability of DNA barcodes is lower using herbarium specimens, compared to freshly collected material, mostly due to lower amplification success, but this is balanced by the increased efficiency of sampling species that have already been collected, identified, and verified by taxonomic experts. The effectiveness of the DNA barcodes for identification (level of discrimination) is assessed using four approaches: the presence of a barcode gap (using pairwise and multiple alignments), formation of monophyletic groups using Neighbour-Joining trees, and sequence similarity in BLASTn searches. These approaches yield similar results, providing relative discrimination levels of 69.4 to 74.9% of all species and 98.6 to 99.8% of genera using both markers. Species discrimination can be further improved using spatially explicit sampling. Mean species discrimination using barcode gap analysis (with a multiple alignment) is 81.6% within 10×10 km squares and 93.3% for 2×2 km squares. Our database of DNA barcodes for Welsh native flowering plants and conifers represents the most complete coverage of any national flora, and offers a valuable platform for a wide range of applications that require accurate species identification. PMID:22701588

WebPrInSeS: automated full-length clone sequence identification and verification using high-throughput sequencing data.

PubMed

Massouras, Andreas; Decouttere, Frederik; Hens, Korneel; Deplancke, Bart

2010-07-01

High-throughput sequencing (HTS) is revolutionizing our ability to obtain cheap, fast and reliable sequence information. Many experimental approaches are expected to benefit from the incorporation of such sequencing features in their pipeline. Consequently, software tools that facilitate such an incorporation should be of great interest. In this context, we developed WebPrInSeS, a web server tool allowing automated full-length clone sequence identification and verification using HTS data. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF) clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one- or two-hybrid assays. Both tools perform de novo assembly using HTS data from any of the three major sequencing platforms. Thus, WebPrInSeS provides a highly integrated, cost-effective and efficient way to sequence-verify or identify clones of interest. WebPrInSeS is available at http://webprinses.epfl.ch/ and is open to all users.

WebPrInSeS: automated full-length clone sequence identification and verification using high-throughput sequencing data

PubMed Central

Massouras, Andreas; Decouttere, Frederik; Hens, Korneel; Deplancke, Bart

2010-01-01

High-throughput sequencing (HTS) is revolutionizing our ability to obtain cheap, fast and reliable sequence information. Many experimental approaches are expected to benefit from the incorporation of such sequencing features in their pipeline. Consequently, software tools that facilitate such an incorporation should be of great interest. In this context, we developed WebPrInSeS, a web server tool allowing automated full-length clone sequence identification and verification using HTS data. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF) clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one- or two-hybrid assays. Both tools perform de novo assembly using HTS data from any of the three major sequencing platforms. Thus, WebPrInSeS provides a highly integrated, cost-effective and efficient way to sequence-verify or identify clones of interest. WebPrInSeS is available at http://webprinses.epfl.ch/ and is open to all users. PMID:20501601

gkmSVM: an R package for gapped-kmer SVM.

PubMed

Ghandi, Mahmoud; Mohammad-Noori, Morteza; Ghareghani, Narges; Lee, Dongwon; Garraway, Levi; Beer, Michael A

2016-07-15

We present a new R package for training gapped-kmer SVM classifiers for DNA and protein sequences. We describe an improved algorithm for kernel matrix calculation that speeds run time by about 2 to 5-fold over our original gkmSVM algorithm. This package supports several sequence kernels, including: gkmSVM, kmer-SVM, mismatch kernel and wildcard kernel. gkmSVM package is freely available through the Comprehensive R Archive Network (CRAN), for Linux, Mac OS and Windows platforms. The C ++ implementation is available at www.beerlab.org/gkmsvm mghandi@gmail.com or mbeer@jhu.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Identification of species by multiplex analysis of variable-length sequences

PubMed Central

Pereira, Filipe; Carneiro, João; Matthiesen, Rune; van Asch, Barbara; Pinto, Nádia; Gusmão, Leonor; Amorim, António

2010-01-01

The quest for a universal and efficient method of identifying species has been a longstanding challenge in biology. Here, we show that accurate identification of species in all domains of life can be accomplished by multiplex analysis of variable-length sequences containing multiple insertion/deletion variants. The new method, called SPInDel, is able to discriminate 93.3% of eukaryotic species from 18 taxonomic groups. We also demonstrate that the identification of prokaryotic and viral species with numeric profiles of fragment lengths is generally straightforward. A computational platform is presented to facilitate the planning of projects and includes a large data set with nearly 1800 numeric profiles for species in all domains of life (1556 for eukaryotes, 105 for prokaryotes and 130 for viruses). Finally, a SPInDel profiling kit for discrimination of 10 mammalian species was successfully validated on highly processed food products with species mixtures and proved to be easily adaptable to multiple screening procedures routinely used in molecular biology laboratories. These results suggest that SPInDel is a reliable and cost-effective method for broad-spectrum species identification that is appropriate for use in suboptimal samples and is amenable to different high-throughput genotyping platforms without the need for DNA sequencing. PMID:20923781

Fundamental Bounds for Sequence Reconstruction from Nanopore Sequencers.

PubMed

Magner, Abram; Duda, Jarosław; Szpankowski, Wojciech; Grama, Ananth

2016-06-01

Nanopore sequencers are emerging as promising new platforms for high-throughput sequencing. As with other technologies, sequencer errors pose a major challenge for their effective use. In this paper, we present a novel information theoretic analysis of the impact of insertion-deletion (indel) errors in nanopore sequencers. In particular, we consider the following problems: (i) for given indel error characteristics and rate, what is the probability of accurate reconstruction as a function of sequence length; (ii) using replicated extrusion (the process of passing a DNA strand through the nanopore), what is the number of replicas needed to accurately reconstruct the true sequence with high probability? Our results provide a number of important insights: (i) the probability of accurate reconstruction of a sequence from a single sample in the presence of indel errors tends quickly (i.e., exponentially) to zero as the length of the sequence increases; and (ii) replicated extrusion is an effective technique for accurate reconstruction. We show that for typical distributions of indel errors, the required number of replicas is a slow function (polylogarithmic) of sequence length - implying that through replicated extrusion, we can sequence large reads using nanopore sequencers. Moreover, we show that in certain cases, the required number of replicas can be related to information-theoretic parameters of the indel error distributions.

Integrating prior knowledge in multiple testing under dependence with applications to detecting differential DNA methylation.

PubMed

Kuan, Pei Fen; Chiang, Derek Y

2012-09-01

DNA methylation has emerged as an important hallmark of epigenetics. Numerous platforms including tiling arrays and next generation sequencing, and experimental protocols are available for profiling DNA methylation. Similar to other tiling array data, DNA methylation data shares the characteristics of inherent correlation structure among nearby probes. However, unlike gene expression or protein DNA binding data, the varying CpG density which gives rise to CpG island, shore and shelf definition provides exogenous information in detecting differential methylation. This article aims to introduce a robust testing and probe ranking procedure based on a nonhomogeneous hidden Markov model that incorporates the above-mentioned features for detecting differential methylation. We revisit the seminal work of Sun and Cai (2009, Journal of the Royal Statistical Society: Series B (Statistical Methodology)71, 393-424) and propose modeling the nonnull using a nonparametric symmetric distribution in two-sided hypothesis testing. We show that this model improves probe ranking and is robust to model misspecification based on extensive simulation studies. We further illustrate that our proposed framework achieves good operating characteristics as compared to commonly used methods in real DNA methylation data that aims to detect differential methylation sites. © 2012, The International Biometric Society.

Application of the CometChip platform to assess DNA damage in field-collected blood samples from turtles.

PubMed

Sykora, Peter; Chiari, Ylenia; Heaton, Andrew; Moreno, Nickolas; Glaberman, Scott; Sobol, Robert W

2018-05-01

DNA damage has been linked to genomic instability and the progressive breakdown of cellular and organismal homeostasis, leading to the onset of disease and reduced longevity. Insults to DNA from endogenous sources include base deamination, base hydrolysis, base alkylation, and metabolism-induced oxidative damage that can lead to single-strand and double-strand DNA breaks. Alternatively, exposure to environmental pollutants, radiation or ultra-violet light, can also contribute to exogenously derived DNA damage. We previously validated a novel, high through-put approach to measure levels of DNA damage in cultured mammalian cells. This new CometChip Platform builds on the classical single cell gel electrophoresis or comet methodology used extensively in environmental toxicology and molecular biology. We asked whether the CometChip Platform could be used to measure DNA damage in samples derived from environmental field studies. To this end, we determined that nucleated erythrocytes from multiple species of turtle could be successfully evaluated in the CometChip Platform to quantify levels of DNA damage. In total, we compared levels of DNA damage in 40 animals from two species: the box turtle (Terrapene carolina) and the red-eared slider (Trachemys scripta elegans). Endogenous levels of DNA damage were identical between the two species, yet we did discover some sex-linked differences and changes in DNA damage accumulation. Based on these results, we confirm that the CometChip Platform allows for the measurement of DNA damage in a large number of samples quickly and accurately, and is particularly adaptable to environmental studies using field-collected samples. Environ. Mol. Mutagen. 59:322-333, 2018. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

[The ENCODE project and functional genomics studies].

PubMed

Ding, Nan; Qu, Hongzhu; Fang, Xiangdong

2014-03-01

Upon the completion of the Human Genome Project, scientists have been trying to interpret the underlying genomic code for human biology. Since 2003, National Human Genome Research Institute (NHGRI) has invested nearly $0.3 billion and gathered over 440 scientists from more than 32 institutions in the United States, China, United Kingdom, Japan, Spain and Singapore to initiate the Encyclopedia of DNA Elements (ENCODE) project, aiming to identify and analyze all regulatory elements in the human genome. Taking advantage of the development of next-generation sequencing technologies and continuous improvement of experimental methods, ENCODE had made remarkable achievements: identified methylation and histone modification of DNA sequences and their regulatory effects on gene expression through altering chromatin structures, categorized binding sites of various transcription factors and constructed their regulatory networks, further revised and updated database for pseudogenes and non-coding RNA, and identified SNPs in regulatory sequences associated with diseases. These findings help to comprehensively understand information embedded in gene and genome sequences, the function of regulatory elements as well as the molecular mechanism underlying the transcriptional regulation by noncoding regions, and provide extensive data resource for life sciences, particularly for translational medicine. We re-viewed the contributions of high-throughput sequencing platform development and bioinformatical technology improve-ment to the ENCODE project, the association between epigenetics studies and the ENCODE project, and the major achievement of the ENCODE project. We also provided our prospective on the role of the ENCODE project in promoting the development of basic and clinical medicine.

Development of a codon optimization strategy using the efor RED reporter gene as a test case

NASA Astrophysics Data System (ADS)

Yip, Chee-Hoo; Yarkoni, Orr; Ajioka, James; Wan, Kiew-Lian; Nathan, Sheila

2018-04-01

Synthetic biology is a platform that enables high-level synthesis of useful products such as pharmaceutically related drugs, bioplastics and green fuels from synthetic DNA constructs. Large-scale expression of these products can be achieved in an industrial compliant host such as Escherichia coli. To maximise the production of recombinant proteins in a heterologous host, the genes of interest are usually codon optimized based on the codon usage of the host. However, the bioinformatics freeware available for standard codon optimization might not be ideal in determining the best sequence for the synthesis of synthetic DNA. Synthesis of incorrect sequences can prove to be a costly error and to avoid this, a codon optimization strategy was developed based on the E. coli codon usage using the efor RED reporter gene as a test case. This strategy replaces codons encoding for serine, leucine, proline and threonine with the most frequently used codons in E. coli. Furthermore, codons encoding for valine and glycine are substituted with the second highly used codons in E. coli. Both the optimized and original efor RED genes were ligated to the pJS209 plasmid backbone using Gibson Assembly and the recombinant DNAs were transformed into E. coli E. cloni 10G strain. The fluorescence intensity per cell density of the optimized sequence was improved by 20% compared to the original sequence. Hence, the developed codon optimization strategy is proposed when designing an optimal sequence for heterologous protein production in E. coli.

FOUNTAIN: A JAVA open-source package to assist large sequencing projects

PubMed Central

Buerstedde, Jean-Marie; Prill, Florian

2001-01-01

Background Better automation, lower cost per reaction and a heightened interest in comparative genomics has led to a dramatic increase in DNA sequencing activities. Although the large sequencing projects of specialized centers are supported by in-house bioinformatics groups, many smaller laboratories face difficulties managing the appropriate processing and storage of their sequencing output. The challenges include documentation of clones, templates and sequencing reactions, and the storage, annotation and analysis of the large number of generated sequences. Results We describe here a new program, named FOUNTAIN, for the management of large sequencing projects . FOUNTAIN uses the JAVA computer language and data storage in a relational database. Starting with a collection of sequencing objects (clones), the program generates and stores information related to the different stages of the sequencing project using a web browser interface for user input. The generated sequences are subsequently imported and annotated based on BLAST searches against the public databases. In addition, simple algorithms to cluster sequences and determine putative polymorphic positions are implemented. Conclusions A simple, but flexible and scalable software package is presented to facilitate data generation and storage for large sequencing projects. Open source and largely platform and database independent, we wish FOUNTAIN to be improved and extended in a community effort. PMID:11591214

Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer.

PubMed

Galamb, Orsolya; Kalmár, Alexandra; Péterfia, Bálint; Csabai, István; Bodor, András; Ribli, Dezső; Krenács, Tibor; Patai, Árpád V; Wichmann, Barnabás; Barták, Barbara Kinga; Tóth, Kinga; Valcz, Gábor; Spisák, Sándor; Tulassay, Zsolt; Molnár, Béla

2016-08-02

The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

Scar-less multi-part DNA assembly design automation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hillson, Nathan J.

The present invention provides a method of a method of designing an implementation of a DNA assembly. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which to assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding flanking homology sequences to each of the DNA oligos. In an exemplary embodiment, the method includes (1) receiving a list of DNA sequence fragments to be assembled together and an order in which tomore » assemble the DNA sequence fragments, (2) designing DNA oligonucleotides (oligos) for each of the DNA sequence fragments, and (3) creating a plan for adding optimized overhang sequences to each of the DNA oligos.« less

Prediction of a rare chromosomal aberration simultaneously with next generation sequencing-based comprehensive chromosome screening in human preimplantation embryos for recurrent pregnancy loss.

PubMed

Lee, Yi-Xuan; Chen, Chien-Wen; Lin, Yi-Hui; Tzeng, Chii-Ruey; Chen, Chi-Huang

2018-01-01

Preimplantation genetic testing has been used widely in recent years as a part of assisted reproductive technology (ART) owing to the breakthrough development of deoxyribonucleic acid (DNA) sequencing. With the advancement of technology and increased resolution of next generation sequencing (NGS), extensive comprehensive chromosome screening along with small clinically significant deletions and duplications can possibly be performed simultaneously. Here, we present a case of rare chromosomal aberrations: 46,XY,dup(15)(q11.2q13),t(16;18)(q23;p11.2), which resulted in a normally developed adult but abnormal gametes leading to recurrent pregnancy loss (RPL). To our best knowledge, this is the first report of t(16;18) translocation with such a small exchanged segment detected by NGS platform of MiSeq system in simultaneous 24-chromosome aneuploidy screening.

Quantifying Genome Editing Outcomes at Endogenous Loci using SMRT Sequencing

PubMed Central

Clark, Joseph; Punjya, Niraj; Sebastiano, Vittorio; Bao, Gang; Porteus, Matthew H

2014-01-01

SUMMARY Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a novel method for quantifying individual genome editing outcomes at any site of interest using single molecule real time (SMRT) DNA sequencing. We show that this approach can be applied at various loci, using multiple engineered nuclease platforms including TALENs, RNA guided endonucleases (CRISPR/Cas9), and ZFNs, and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach facilitates the evaluation of new gene editing technologies and permits sensitive quantification of editing outcomes in almost every experimental system used. PMID:24685129

Electrochemical label-free and sensitive nanobiosensing of DNA hybridization by graphene oxide modified pencil graphite electrode.

PubMed

Ahour, F; Shamsi, A

2017-09-01

Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π- π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM-0.5 μM with a detection limit of 4.3 × 10 -11  M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future. Copyright © 2017 Elsevier Inc. All rights reserved.

Optimization of techniques for multiple platform testing in small, precious samples such as human chorionic villus sampling.

PubMed

Pisarska, Margareta D; Akhlaghpour, Marzieh; Lee, Bora; Barlow, Gillian M; Xu, Ning; Wang, Erica T; Mackey, Aaron J; Farber, Charles R; Rich, Stephen S; Rotter, Jerome I; Chen, Yii-der I; Goodarzi, Mark O; Guller, Seth; Williams, John

2016-11-01

Multiple testing to understand global changes in gene expression based on genetic and epigenetic modifications is evolving. Chorionic villi, obtained for prenatal testing, is limited, but can be used to understand ongoing human pregnancies. However, optimal storage, processing and utilization of CVS for multiple platform testing have not been established. Leftover CVS samples were flash-frozen or preserved in RNAlater. Modifications to standard isolation kits were performed to isolate quality DNA and RNA from samples as small as 2-5 mg. RNAlater samples had significantly higher RNA yields and quality and were successfully used in microarray and RNA-sequencing (RNA-seq). RNA-seq libraries generated using 200 versus 800-ng RNA showed similar biological coefficients of variation. RNAlater samples had lower DNA yields and quality, which improved by heating the elution buffer to 70 °C. Purification of DNA was not necessary for bisulfite-conversion and genome-wide methylation profiling. CVS cells were propagated and continue to express genes found in freshly isolated chorionic villi. CVS samples preserved in RNAlater are superior. Our optimized techniques provide specimens for genetic, epigenetic and gene expression studies from a single small sample which can be used to develop diagnostics and treatments using a systems biology approach in the prenatal period. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

Complete chloroplast and ribosomal sequences for 30 accessions elucidate evolution of Oryza AA genome species

PubMed Central

Kim, Kyunghee; Lee, Sang-Choon; Lee, Junki; Yu, Yeisoo; Yang, Kiwoung; Choi, Beom-Soon; Koh, Hee-Jong; Waminal, Nomar Espinosa; Choi, Hong-Il; Kim, Nam-Hoon; Jang, Woojong; Park, Hyun-Seung; Lee, Jonghoon; Lee, Hyun Oh; Joh, Ho Jun; Lee, Hyeon Ju; Park, Jee Young; Perumal, Sampath; Jayakodi, Murukarthick; Lee, Yun Sun; Kim, Backki; Copetti, Dario; Kim, Soonok; Kim, Sunggil; Lim, Ki-Byung; Kim, Young-Dong; Lee, Jungho; Cho, Kwang-Su; Park, Beom-Seok; Wing, Rod A.; Yang, Tae-Jin

2015-01-01

Cytoplasmic chloroplast (cp) genomes and nuclear ribosomal DNA (nR) are the primary sequences used to understand plant diversity and evolution. We introduce a high-throughput method to simultaneously obtain complete cp and nR sequences using Illumina platform whole-genome sequence. We applied the method to 30 rice specimens belonging to nine Oryza species. Concurrent phylogenomic analysis using cp and nR of several of specimens of the same Oryza AA genome species provides insight into the evolution and domestication of cultivated rice, clarifying three ambiguous but important issues in the evolution of wild Oryza species. First, cp-based trees clearly classify each lineage but can be biased by inter-subspecies cross-hybridization events during speciation. Second, O. glumaepatula, a South American wild rice, includes two cytoplasm types, one of which is derived from a recent interspecies hybridization with O. longistminata. Third, the Australian O. rufipogan-type rice is a perennial form of O. meridionalis. PMID:26506948

CNV-seq, a new method to detect copy number variation using high-throughput sequencing.

PubMed

Xie, Chao; Tammi, Martti T

2009-03-06

DNA copy number variation (CNV) has been recognized as an important source of genetic variation. Array comparative genomic hybridization (aCGH) is commonly used for CNV detection, but the microarray platform has a number of inherent limitations. Here, we describe a method to detect copy number variation using shotgun sequencing, CNV-seq. The method is based on a robust statistical model that describes the complete analysis procedure and allows the computation of essential confidence values for detection of CNV. Our results show that the number of reads, not the length of the reads is the key factor determining the resolution of detection. This favors the next-generation sequencing methods that rapidly produce large amount of short reads. Simulation of various sequencing methods with coverage between 0.1x to 8x show overall specificity between 91.7 - 99.9%, and sensitivity between 72.2 - 96.5%. We also show the results for assessment of CNV between two individual human genomes.

PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

PubMed

Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

2013-06-14

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

AQME: A forensic mitochondrial DNA analysis tool for next-generation sequencing data.

PubMed

Sturk-Andreaggi, Kimberly; Peck, Michelle A; Boysen, Cecilie; Dekker, Patrick; McMahon, Timothy P; Marshall, Charla K

2017-11-01

The feasibility of generating mitochondrial DNA (mtDNA) data has expanded considerably with the advent of next-generation sequencing (NGS), specifically in the generation of entire mtDNA genome (mitogenome) sequences. However, the analysis of these data has emerged as the greatest challenge to implementation in forensics. To address this need, a custom toolkit for use in the CLC Genomics Workbench (QIAGEN, Hilden, Germany) was developed through a collaborative effort between the Armed Forces Medical Examiner System - Armed Forces DNA Identification Laboratory (AFMES-AFDIL) and QIAGEN Bioinformatics. The AFDIL-QIAGEN mtDNA Expert, or AQME, generates an editable mtDNA profile that employs forensic conventions and includes the interpretation range required for mtDNA data reporting. AQME also integrates an mtDNA haplogroup estimate into the analysis workflow, which provides the analyst with phylogenetic nomenclature guidance and a profile quality check without the use of an external tool. Supplemental AQME outputs such as nucleotide-per-position metrics, configurable export files, and an audit trail are produced to assist the analyst during review. AQME is applied to standard CLC outputs and thus can be incorporated into any mtDNA bioinformatics pipeline within CLC regardless of sample type, library preparation or NGS platform. An evaluation of AQME was performed to demonstrate its functionality and reliability for the analysis of mitogenome NGS data. The study analyzed Illumina mitogenome data from 21 samples (including associated controls) of varying quality and sample preparations with the AQME toolkit. A total of 211 tool edits were automatically applied to 130 of the 698 total variants reported in an effort to adhere to forensic nomenclature. Although additional manual edits were required for three samples, supplemental tools such as mtDNA haplogroup estimation assisted in identifying and guiding these necessary modifications to the AQME-generated profile. Along with profile generation, AQME reported accurate haplogroups for 18 of the 19 samples analyzed. The single errant haplogroup assignment, although phylogenetically close, identified a bug that only affects partial mitogenome data. Future adjustments to AQME's haplogrouping tool will address this bug as well as enhance the overall scoring strategy to better refine and automate haplogroup assignments. As NGS enables broader use of the mtDNA locus in forensics, the availability of AQME and other forensic-focused mtDNA analysis tools will ease the transition and further support mitogenome analysis within routine casework. Toward this end, the AFMES-AFDIL has utilized the AQME toolbox in conjunction with the CLC Genomics Workbench to successfully validate and implement two NGS mitogenome methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Voltammetric behaviour of free DNA bases, methylcytosine and oligonucleotides at disposable screen printed graphite electrode platforms.

PubMed

Brotons, Ariadna; Mas, Luis Alcaraz; Metters, Jonathan P; Banks, Craig E; Iniesta, Jesús

2013-09-21

Improvements in analytical methods for the determination and quantification of methylcytosine in DNA are vital since it has the potential to be used as a biomarker to detect different diseases in the first stage such as in the case of carcinomas and sterility. In this work we utilized screen printed graphite electrodes (SPGE) for studying the electrochemical response of all free DNA bases, methylcytosine and short oligonucleotides by cyclic voltammetry (CV) and square wave voltammetry (SWV). CV and SWV responses of free DNA bases and methylcytosine have been investigated by using SPGE platforms and the feasibility of detecting and quantifying cytosine and methylcytosine as free DNA moieties has been evaluated as a function of pH, concentration and the presence of the other free DNA bases in solution simultaneously. Repeatability of using SWV has been performed for the electrochemical behavior of both 250 μM cytosine and 250 μM methylcytosine in the presence of 25 μM guanine, with coefficient of variations of 6.9% and 2.6% respectively based upon peak current (N = 5). Six-mer oligonucleotides with a sequence 5'-XXXCGC-3', where the XXX motif corresponds to TTT, TTA, TAA and AAA have been performed using SWV in 0.1 M acetate buffer pH 5.0 to explore how the DNA base position effects the electrooxidation of guanine and cytosine into the oligonucleotide. Furthermore SWV comparisons of the electrooxidation of the oligonucleotides 5'-CGCGCG-3' and its methylated 5'-mCGmCGmCG-3' have been performed with concentrations in acetate buffer solutions, and the interaction of both oligonucleotides with the graphitic surface of the SPGE has been demonstrated by fitting well-known adsorption models such as Freundlich and Langmuir kinetics according to the SWV current response of guanine, cytosine and methylcytosine into the oligonucleotide.

Aptamer-based impedimetric sensor for bacterial typing.

PubMed

Labib, Mahmoud; Zamay, Anna S; Kolovskaya, Olga S; Reshetneva, Irina T; Zamay, Galina S; Kibbee, Richard J; Sattar, Syed A; Zamay, Tatiana N; Berezovski, Maxim V

2012-10-02

The development of an aptamer-based impedimetric sensor for typing of bacteria (AIST-B) is presented. Highly specific DNA aptamers to Salmonella enteritidis were selected via Cell-SELEX technique. Twelve rounds of selection were performed; each comprises a positive selection step against S. enteritidis and a negative selection step against a mixture of related pathogens, including Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Citrobacter freundii, to ensure the species-specificity of the selected aptamers. After sequencing of the pool showing the highest binding affinity to S. enteritidis, a DNA sequence of high affinity to the bacteria was integrated into an impedimetric sensor via self-assembly onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Remarkably, this aptasensor is highly selective and can successfully detect S. enteritidis down to 600 CFU mL(-1) (equivalent to 18 CFU in 30 μL assay volume) in 10 min and distinguish it from other Salmonella species, including S. typhimurium and S. choleraesuis. This report is envisaged to open a new venue for the aptamer-based typing of a variety of microorganisms using a rapid, economic, and label-free electrochemical platform.

Amplified electrochemical detection of nucleic acid hybridization via selective preconcentration of unmodified gold nanoparticles.

PubMed

Li, Yuan; Tian, Rui; Zheng, Xingwang; Huang, Rongfu

2016-08-31

The common drawback of optical methods for rapid detection of nucleic acid by exploiting the differential affinity of single-/double-stranded nucleic acids for unmodified gold nanoparticles (AuNPs) is its relatively low sensitivity. In this article, on the basis of selective preconcentration of AuNPs unprotected by single-stranded DNA (ssDNA) binding, a novel electrochemical strategy for nucleic acid sequence identification assay has been developed. Through detecting the redox signal mediated by AuNPs on 1, 6-hexanedithiol blocked gold electrode, the proposed method is able to ensure substantial signal amplification and a low background current. This strategy is demonstrated for quantitative analysis of the target microRNA (let-7a) in human breast adenocarcinoma cells, and a detection limit of 16 fM is readily achieved with desirable specificity and sensitivity. These results indicate that the selective preconcentration of AuNPs for electrochemical signal readout can offer a promising platform for the detection of specific nucleic acid sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

Disruption of SMIM1 causes the Vel− blood type

PubMed Central

Ballif, Bryan A; Helias, Virginie; Peyrard, Thierry; Menanteau, Cécile; Saison, Carole; Lucien, Nicole; Bourgouin, Sébastien; Le Gall, Maude; Cartron, Jean-Pierre; Arnaud, Lionel

2013-01-01

Here, we report the biochemical and genetic basis of the Vel blood group antigen, which has been a vexing mystery for decades, especially as anti-Vel regularly causes severe haemolytic transfusion reactions. The protein carrying the Vel blood group antigen was biochemically purified from red blood cell membranes. Mass spectrometry-based de novo peptide sequencing identified this protein to be small integral membrane protein 1 (SMIM1), a previously uncharacterized single-pass membrane protein. Expression of SMIM1 cDNA in Vel− cultured cells generated anti-Vel cell surface reactivity, confirming that SMIM1 encoded the Vel blood group antigen. A cohort of 70 Vel− individuals was found to be uniformly homozygous for a 17 nucleotide deletion in the coding sequence of SMIM1. The genetic homogeneity of the Vel− blood type, likely having a common origin, facilitated the development of two highly specific DNA-based tests for rapid Vel genotyping, which can be easily integrated into blood group genotyping platforms. These results answer a 60-year-old riddle and provide tools of immediate assistance to all clinicians involved in the care of Vel− patients. PMID:23505126

Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

DOEpatents

Gardner, Shea N; Mariella, Jr., Raymond P; Christian, Allen T; Young, Jennifer A; Clague, David S

2013-06-25

A method of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths.

Electronic Properties of Synthetic Shrimp Pathogens-derived DNA Schottky Diodes.

PubMed

Rizan, Nastaran; Yew, Chan Yen; Niknam, Maryam Rajabpour; Krishnasamy, Jegenathan; Bhassu, Subha; Hong, Goh Zee; Devadas, Sridevi; Din, Mohamed Shariff Mohd; Tajuddin, Hairul Anuar; Othman, Rofina Yasmin; Phang, Siew Moi; Iwamoto, Mitsumasa; Periasamy, Vengadesh

2018-01-17

The exciting discovery of the semiconducting-like properties of deoxyribonucleic acid (DNA) and its potential applications in molecular genetics and diagnostics in recent times has resulted in a paradigm shift in biophysics research. Recent studies in our laboratory provide a platform towards detecting charge transfer mechanism and understanding the electronic properties of DNA based on the sequence-specific electronic response, which can be applied as an alternative to identify or detect DNA. In this study, we demonstrate a novel method for identification of DNA from different shrimp viruses and bacteria using electronic properties of DNA obtained from both negative and positive bias regions in current-voltage (I-V) profiles. Characteristic electronic properties were calculated and used for quantification and further understanding in the identification process. Aquaculture in shrimp industry is a fast-growing food sector throughout the world. However, shrimp culture in many Asian countries faced a huge economic loss due to disease outbreaks. Scientists have been using specific established methods for detecting shrimp infection, but those methods do have their significant drawbacks due to many inherent factors. As such, we believe that this simple, rapid, sensitive and cost-effective tool can be used for detection and identification of DNA from different shrimp viruses and bacteria.

Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions

DOEpatents

Gardner, Shea N [San Leandro, CA; Mariella, Jr., Raymond P.; Christian, Allen T [Tracy, CA; Young, Jennifer A [Berkeley, CA; Clague, David S [Livermore, CA

2011-01-18

A method of fabricating a DNA molecule of user-defined sequence. The method comprises the steps of preselecting a multiplicity of DNA sequence segments that will comprise the DNA molecule of user-defined sequence, separating the DNA sequence segments temporally, and combining the multiplicity of DNA sequence segments with at least one polymerase enzyme wherein the multiplicity of DNA sequence segments join to produce the DNA molecule of user-defined sequence. Sequence segments may be of length n, where n is an even or odd integer. In one embodiment the length of desired hybridizing overlap is specified by the user and the sequences and the protocol for combining them are guided by computational (bioinformatics) predictions. In one embodiment sequence segments are combined from multiple reading frames to span the same region of a sequence, so that multiple desired hybridizations may occur with different overlap lengths. In one embodiment starting sequence fragments are of different lengths, n, n+1, n+2, etc.

Comprehensive comparison of three commercial human whole-exome capture platforms.

PubMed

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.

Protein alignment algorithms with an efficient backtracking routine on multiple GPUs.

PubMed

Blazewicz, Jacek; Frohmberg, Wojciech; Kierzynka, Michal; Pesch, Erwin; Wojciechowski, Pawel

2011-05-20

Pairwise sequence alignment methods are widely used in biological research. The increasing number of sequences is perceived as one of the upcoming challenges for sequence alignment methods in the nearest future. To overcome this challenge several GPU (Graphics Processing Unit) computing approaches have been proposed lately. These solutions show a great potential of a GPU platform but in most cases address the problem of sequence database scanning and computing only the alignment score whereas the alignment itself is omitted. Thus, the need arose to implement the global and semiglobal Needleman-Wunsch, and Smith-Waterman algorithms with a backtracking procedure which is needed to construct the alignment. In this paper we present the solution that performs the alignment of every given sequence pair, which is a required step for progressive multiple sequence alignment methods, as well as for DNA recognition at the DNA assembly stage. Performed tests show that the implementation, with performance up to 6.3 GCUPS on a single GPU for affine gap penalties, is very efficient in comparison to other CPU and GPU-based solutions. Moreover, multiple GPUs support with load balancing makes the application very scalable. The article shows that the backtracking procedure of the sequence alignment algorithms may be designed to fit in with the GPU architecture. Therefore, our algorithm, apart from scores, is able to compute pairwise alignments. This opens a wide range of new possibilities, allowing other methods from the area of molecular biology to take advantage of the new computational architecture. Performed tests show that the efficiency of the implementation is excellent. Moreover, the speed of our GPU-based algorithms can be almost linearly increased when using more than one graphics card.

High resolution melting analytical platform for rapid prenatal and postnatal diagnosis of β-thalassemia common among Southeast Asian population.

PubMed

Prajantasen, Thanet; Fucharoen, Supan; Fucharoen, Goonnapa

2015-02-20

High resolution melting (HRM) analysis is a powerful technology for scanning sequence alteration. We have applied this HRM assay to screen common β-thalassemia mutations found among Southeast Asian population. Known DNA samples with 8 common mutations were used in initial development of the methods including -28 A-G, codon 17 A-T, IVSI-1G-T, IVSI-5G-C, codon 26G-A (Hb E), codons 41/42 -TTCT, codons 71/72+A and IVSII-654 C-T. Further validation was done on 60 postnatal and 6 prenatal diagnoses of β-thalassemia. Each mutation has specific HRM profile which could be used in rapid screening. Apart from those with DNA deletions, the results of HRM assay matched 100% with those of routine diagnosis made by routine allele specific PCR. In addition, the HRM assay could initially recognize three unknown mutations including a hitherto un-described one in Thai population. The established HRM assay should prove useful for rapid and high throughput platform for screening and prenatal diagnosis of β-thalassemia common in the region. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification and characterization of EBV genomes in spontaneously immortalized human peripheral blood B lymphocytes by NGS technology.

PubMed

Lei, Haiyan; Li, Tianwei; Hung, Guo-Chiuan; Li, Bingjie; Tsai, Shien; Lo, Shyh-Ching

2013-11-19

We conducted genomic sequencing to identify Epstein Barr Virus (EBV) genomes in 2 human peripheral blood B lymphocytes that underwent spontaneous immortalization promoted by mycoplasma infections in culture, using the high-throughput sequencing (HTS) Illumina MiSeq platform. The purpose of this study was to examine if rapid detection and characterization of a viral agent could be effectively achieved by HTS using a platform that has become readily available in general biology laboratories. Raw read sequences, averaging 175 bps in length, were mapped with DNA databases of human, bacteria, fungi and virus genomes using the CLC Genomics Workbench bioinformatics tool. Overall 37,757 out of 49,520,834 total reads in one lymphocyte line (# K4413-Mi) and 28,178 out of 45,335,960 reads in the other lymphocyte line (# K4123-Mi) were identified as EBV sequences. The two EBV genomes with estimated 35.22-fold and 31.06-fold sequence coverage respectively, designated K4413-Mi EBV and K4123-Mi EBV (GenBank accession number KC440852 and KC440851 respectively), are characteristic of type-1 EBV. Sequence comparison and phylogenetic analysis among K4413-Mi EBV, K4123-Mi EBV and the EBV genomes previously reported to GenBank as well as the NA12878 EBV genome assembled from database of the 1000 Genome Project showed that these 2 EBVs are most closely related to B95-8, an EBV previously isolated from a patient with infectious mononucleosis and WT-EBV. They are less similar to EBVs associated with nasopharyngeal carcinoma (NPC) from Hong Kong and China as well as the Akata strain of a case of Burkitt's lymphoma from Japan. They are most different from type 2 EBV found in Western African Burkitt's lymphoma.

Sockeye: A 3D Environment for Comparative Genomics

PubMed Central

Montgomery, Stephen B.; Astakhova, Tamara; Bilenky, Mikhail; Birney, Ewan; Fu, Tony; Hassel, Maik; Melsopp, Craig; Rak, Marcin; Robertson, A. Gordon; Sleumer, Monica; Siddiqui, Asim S.; Jones, Steven J.M.

2004-01-01

Comparative genomics techniques are used in bioinformatics analyses to identify the structural and functional properties of DNA sequences. As the amount of available sequence data steadily increases, the ability to perform large-scale comparative analyses has become increasingly relevant. In addition, the growing complexity of genomic feature annotation means that new approaches to genomic visualization need to be explored. We have developed a Java-based application called Sockeye that uses three-dimensional (3D) graphics technology to facilitate the visualization of annotation and conservation across multiple sequences. This software uses the Ensembl database project to import sequence and annotation information from several eukaryotic species. A user can additionally import their own custom sequence and annotation data. Individual annotation objects are displayed in Sockeye by using custom 3D models. Ensembl-derived and imported sequences can be analyzed by using a suite of multiple and pair-wise alignment algorithms. The results of these comparative analyses are also displayed in the 3D environment of Sockeye. By using the Java3D API to visualize genomic data in a 3D environment, we are able to compactly display cross-sequence comparisons. This provides the user with a novel platform for visualizing and comparing genomic feature organization. PMID:15123592

Skin Microbiome Surveys Are Strongly Influenced by Experimental Design.

PubMed

Meisel, Jacquelyn S; Hannigan, Geoffrey D; Tyldsley, Amanda S; SanMiguel, Adam J; Hodkinson, Brendan P; Zheng, Qi; Grice, Elizabeth A

2016-05-01

Culture-independent studies to characterize skin microbiota are increasingly common, due in part to affordable and accessible sequencing and analysis platforms. Compared to culture-based techniques, DNA sequencing of the bacterial 16S ribosomal RNA (rRNA) gene or whole metagenome shotgun (WMS) sequencing provides more precise microbial community characterizations. Most widely used protocols were developed to characterize microbiota of other habitats (i.e., gastrointestinal) and have not been systematically compared for their utility in skin microbiome surveys. Here we establish a resource for the cutaneous research community to guide experimental design in characterizing skin microbiota. We compare two widely sequenced regions of the 16S rRNA gene to WMS sequencing for recapitulating skin microbiome community composition, diversity, and genetic functional enrichment. We show that WMS sequencing most accurately recapitulates microbial communities, but sequencing of hypervariable regions 1-3 of the 16S rRNA gene provides highly similar results. Sequencing of hypervariable region 4 poorly captures skin commensal microbiota, especially Propionibacterium. WMS sequencing, which is resource and cost intensive, provides evidence of a community's functional potential; however, metagenome predictions based on 16S rRNA sequence tags closely approximate WMS genetic functional profiles. This study highlights the importance of experimental design for downstream results in skin microbiome surveys. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Skin microbiome surveys are strongly influenced by experimental design

PubMed Central

Meisel, Jacquelyn S.; Hannigan, Geoffrey D.; Tyldsley, Amanda S.; SanMiguel, Adam J.; Hodkinson, Brendan P.; Zheng, Qi; Grice, Elizabeth A.

2016-01-01

Culture-independent studies to characterize skin microbiota are increasingly common, due in part to affordable and accessible sequencing and analysis platforms. Compared to culture-based techniques, DNA sequencing of the bacterial 16S ribosomal RNA (rRNA) gene or whole metagenome shotgun (WMS) sequencing provide more precise microbial community characterizations. Most widely used protocols were developed to characterize microbiota of other habitats (i.e. gastrointestinal), and have not been systematically compared for their utility in skin microbiome surveys. Here we establish a resource for the cutaneous research community to guide experimental design in characterizing skin microbiota. We compare two widely sequenced regions of the 16S rRNA gene to WMS sequencing for recapitulating skin microbiome community composition, diversity, and genetic functional enrichment. We show that WMS sequencing most accurately recapitulates microbial communities, but sequencing of hypervariable regions 1-3 of the 16S rRNA gene provides highly similar results. Sequencing of hypervariable region 4 poorly captures skin commensal microbiota, especially Propionibacterium. WMS sequencing, which is resource- and cost-intensive, provides evidence of a community’s functional potential; however, metagenome predictions based on 16S rRNA sequence tags closely approximate WMS genetic functional profiles. This work highlights the importance of experimental design for downstream results in skin microbiome surveys. PMID:26829039

Variations in 5S rDNAs in diploid and tetraploid offspring of red crucian carp × common carp.

PubMed

Ye, Lihai; Zhang, Chun; Tang, Xiaojun; Chen, Yiyi; Liu, Shaojun

2017-08-08

The allotetraploid hybrid fish (4nAT) that was created in a previous study through an intergeneric cross between red crucian carp (Carassius auratus red var., ♀) and common carp (Cyprinus carpio L., ♂) provided an excellent platform to investigate the effect of hybridization and polyploidization on the evolution of 5S rDNA. The 5S rDNAs of paternal common carp were made up of a coding sequence (CDS) and a non-transcribed spacer (NTS) unit, and while the 5S rDNAs of maternal red crucian carp contained a CDS and a NTS unit, they also contained a variable number of interposed regions (IPRs). The CDSs of the 5S rDNAs in both parental fishes were conserved, while their NTS units seemed to have been subjected to rapid evolution. The diploid hybrid 2nF 1 inherited all the types of 5S rDNAs in both progenitors and there were no signs of homeologous recombination in the 5S rDNAs of 2nF 1 by sequencing of PCR products. We obtained two segments of 5S rDNA with a total length of 16,457 bp from allotetraploid offspring 4nAT through bacterial artificial chromosome (BAC) sequencing. Using this sequence together with the 5S rDNA sequences amplified from the genomic DNA of 4nAT, we deduced that the 5S rDNAs of 4nAT might be inherited from the maternal progenitor red crucian carp. Additionally, the IPRs in the 5S rDNAs of 4nAT contained A-repeats and TA-repeats, which was not the case for the IPRs in the 5S rDNAs of 2nF 1 . We also detected two signals of a 200-bp fragment of 5S rDNA in the chromosomes of parental progenitors and hybrid progenies by fluorescence in situ hybridization (FISH). We deduced that during the evolution of 5S rDNAs in different ploidy hybrid fishes, interlocus gene conversion events and tandem repeat insertion events might occurred in the process of polyploidization. This study provided new insights into the relationship among the evolution of 5S rDNAs, hybridization and polyploidization, which were significant in clarifying the genome evolution of polyploid fish.

Highly sensitive dual mode electrochemical platform for microRNA detection

NASA Astrophysics Data System (ADS)

Jolly, Pawan; Batistuti, Marina R.; Miodek, Anna; Zhurauski, Pavel; Mulato, Marcelo; Lindsay, Mark A.; Estrela, Pedro

2016-11-01

MicroRNAs (miRNAs) play crucial regulatory roles in various human diseases including cancer, making them promising biomarkers. However, given the low levels of miRNAs present in blood, their use as cancer biomarkers requires the development of simple and effective analytical methods. Herein, we report the development of a highly sensitive dual mode electrochemical platform for the detection of microRNAs. The platform was developed using peptide nucleic acids as probes on gold electrode surfaces to capture target miRNAs. A simple amplification strategy using gold nanoparticles has been employed exploiting the inherent charges of the nucleic acids. Electrochemical impedance spectroscopy was used to monitor the changes in capacitance upon any binding event, without the need for any redox markers. By using thiolated ferrocene, a complementary detection mode on the same sensor was developed where the increasing peaks of ferrocene were recorded using square wave voltammetry with increasing miRNA concentration. This dual-mode approach allows detection of miRNA with a limit of detection of 0.37 fM and a wide dynamic range from 1 fM to 100 nM along with clear distinction from mismatched target miRNA sequences. The electrochemical platform developed can be easily expanded to other miRNA/DNA detection along with the development of microarray platforms.

Draft genome sequence of Enterobacter cloacae HBY, a ST128 clinical strain co-producing KPC-2 and NDM-1 carbapenemases.

PubMed

Li, Xi; Zhu, Yongze; Shen, Mengyuan; Du, Jing; Zhang, Lei; Wang, Dairong

2018-03-01

Enterobacter cloacae is one of the major pathogens responsible for a variety of human infections. Here we report the draft genome sequence of multidrug-resistant E. cloacae strain HBY isolated from a female patient in China. Whole genomic DNA of E. cloacae strain HBY was extracted and was sequenced using an Illumina HiSeq™ 2000 platform. The generated sequence reads were assembled using CLC Genomics Workbench. The draft genome was annotated using Rapid Annotations using Subsystems Technology (RAST), and the presence of antimicrobial resistance genes was identified. The 5799439-bp genome contains various antimicrobial resistance genes conferring resistance to aminoglycosides, β-lactams, fosfomycin, macrolides, sulphonamides and fluoroquinolones. Notably, the strain was identified to carry two main carbapenemase genes (bla KPC-2 and bla NDM-1 ). The genome sequence reported in this study will provide valuable information to understand antibiotic resistance mechanisms in this strain. It is important to monitor the spread strains of Enterobacter sp. encoding both of these carbapenemase genes. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Transcriptome sequencing analysis of novel sRNAs of Kineococcus radiotolerans in response to ionizing radiation.

PubMed

Chen, Zhouwei; Li, Lufeng; Shan, Zhan; Huang, Hannian; Chen, Huan; Ding, Xianfeng; Guo, Jiangfeng; Liu, Lili

2016-11-01

Kineococcus radiotolerans is a Gram-positive, radio-resistant bacterium isolated from a radioactive environment. The small noncoding RNAs (sRNAs) in bacteria are reported to play roles in the immediate response to stress and/or the recovery from stress. The analysis of K. radiotolerans transcriptome sequencing results can identify these sRNAs in a genome-wide detection, using RNA sequencing (RNA-seq) by the deep sequencing technique. In this study, the raw data of radiation-exposed samples (RS) and control samples (CS) were acquired separately from the sequencing platform. There were 217 common sRNA candidates in the two samples screened in the genome-wide scale by bioinformatics analysis. There were 43 differentially expressed sRNA candidates, including 28 up-regulated and 15 down-regulated ones. The down-regulated sRNAs were selected for the sRNA target prediction, of which 12 sRNAs that may modulate the genes related to the transcription regulation and DNA repair were considered as the candidates involved in the radio-resistance regulation system. Copyright © 2016 Elsevier GmbH. All rights reserved.

PHYLOViZ: phylogenetic inference and data visualization for sequence based typing methods

PubMed Central

2012-01-01

Background With the decrease of DNA sequencing costs, sequence-based typing methods are rapidly becoming the gold standard for epidemiological surveillance. These methods provide reproducible and comparable results needed for a global scale bacterial population analysis, while retaining their usefulness for local epidemiological surveys. Online databases that collect the generated allelic profiles and associated epidemiological data are available but this wealth of data remains underused and are frequently poorly annotated since no user-friendly tool exists to analyze and explore it. Results PHYLOViZ is platform independent Java software that allows the integrated analysis of sequence-based typing methods, including SNP data generated from whole genome sequence approaches, and associated epidemiological data. goeBURST and its Minimum Spanning Tree expansion are used for visualizing the possible evolutionary relationships between isolates. The results can be displayed as an annotated graph overlaying the query results of any other epidemiological data available. Conclusions PHYLOViZ is a user-friendly software that allows the combined analysis of multiple data sources for microbial epidemiological and population studies. It is freely available at http://www.phyloviz.net. PMID:22568821

Microsecond Simulations of DNA and Ion Transport in Nanopores with Novel Ion-Ion and Ion-Nucleotides Effective Potentials

PubMed Central

De Biase, Pablo M.; Markosyan, Suren; Noskov, Sergei

2014-01-01

We developed a novel scheme based on the Grand-Canonical Monte-Carlo/Brownian Dynamics (GCMC/BD) simulations and have extended it to studies of ion currents across three nanopores with the potential for ssDNA sequencing: solid-state nanopore Si3N4, α-hemolysin, and E111N/M113Y/K147N mutant. To describe nucleotide-specific ion dynamics compatible with ssDNA coarse-grained model, we used the Inverse Monte-Carlo protocol, which maps the relevant ion-nucleotide distribution functions from an all-atom MD simulations. Combined with the previously developed simulation platform for Brownian Dynamic (BD) simulations of ion transport, it allows for microsecond- and millisecond-long simulations of ssDNA dynamics in nanopore with a conductance computation accuracy that equals or exceeds that of all-atom MD simulations. In spite of the simplifications, the protocol produces results that agree with the results of previous studies on ion conductance across open channels and provide direct correlations with experimentally measured blockade currents and ion conductances that have been estimated from all-atom MD simulations. PMID:24738152

Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore

NASA Astrophysics Data System (ADS)

Perera, Rukshan T.; Fleming, Aaron M.; Johnson, Robert P.; Burrows, Cynthia J.; White, Henry S.

2015-02-01

The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2‧-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5‧ or 3‧ directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

Electrochemical sensing and biosensing platform based on chemically reduced graphene oxide.

PubMed

Zhou, Ming; Zhai, Yueming; Dong, Shaojun

2009-07-15

In this paper, the characterization and application of a chemically reduced graphene oxide modified glassy carbon (CR-GO/GC) electrode, a novel electrode system, for the preparation of electrochemical sensing and biosensing platform are proposed. Different kinds of important inorganic and organic electroactive compounds (i.e., probe molecule (potassium ferricyanide), free bases of DNA (guanine (G), adenine (A), thymine (T), and cytosine (C)), oxidase/dehydrogenase-related molecules (hydrogen peroxide (H2O2)/beta-nicotinamide adenine dinucleotide (NADH)), neurotransmitters (dopamine (DA)), and other biological molecules (ascorbic acid (AA), uric acid (UA), and acetaminophen (APAP)) were employed to study their electrochemical responses at the CR-GO/GC electrode, which shows more favorable electron transfer kinetics than graphite modified glassy carbon (graphite/GC) and glassy carbon (GC) electrodes. The greatly enhanced electrochemical reactivity of the four free bases of DNA at the CR-GO/GC electrode compared with that at graphite/GC and GC electrodes makes the CR-GO/GC electrode a better choice for the electrochemical biosensing of four DNA bases in both the single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) at physiological pH without a prehydrolysis step. This allows us to detect a single-nucleotide polymorphism (SNP) site for short oligomers with a particular sequence at the CR-GO/GC electrode without any hybridization or labeling processes in this work, suggesting the potential applications of CR-GO in the label-free electrochemical detection of DNA hybridization or DNA damage for further research. Based on the greatly enhanced electrochemical reactivity of H2O2 and NADH at the CR-GO/GC electrode, CR-GO/GC electrode-based bioelectrodes (in connection with glucose oxidase (GOD) and alcohol dehydrogenase (ADH)) show a better analytical performance for the detection of glucose and ethanol compared with graphite/GC- or GC-based bioelectrodes. By comparing the electrochemical performance of CR-GO with that of the conventional graphite and GC, we reveal that CR-GO with the nature of a single sheet showing favorable electrochemical activity should be a kind of more robust and advanced carbon electrode material which may hold great promise for electrochemical sensors and biosensors design.

SG-ADVISER mtDNA: a web server for mitochondrial DNA annotation with data from 200 samples of a healthy aging cohort.

PubMed

Rueda, Manuel; Torkamani, Ali

2017-08-18

Whole genome and exome sequencing usually include reads containing mitochondrial DNA (mtDNA). Yet, state-of-the-art pipelines and services for human nuclear genome variant calling and annotation do not handle mitochondrial genome data appropriately. As a consequence, any researcher desiring to add mtDNA variant analysis to their investigations is forced to explore the literature for mtDNA pipelines, evaluate them, and implement their own instance of the desired tool. This task is far from trivial, and can be prohibitive for non-bioinformaticians. We have developed SG-ADVISER mtDNA, a web server to facilitate the analysis and interpretation of mtDNA genomic data coming from next generation sequencing (NGS) experiments. The server was built in the context of our SG-ADVISER framework and on top of the MtoolBox platform (Calabrese et al., Bioinformatics 30(21):3115-3117, 2014), and includes most of its functionalities (i.e., assembly of mitochondrial genomes, heteroplasmic fractions, haplogroup assignment, functional and prioritization analysis of mitochondrial variants) as well as a back-end and a front-end interface. The server has been tested with unpublished data from 200 individuals of a healthy aging cohort (Erikson et al., Cell 165(4):1002-1011, 2016) and their data is made publicly available here along with a preliminary analysis of the variants. We observed that individuals over ~90 years old carried low levels of heteroplasmic variants in their genomes. SG-ADVISER mtDNA is a fast and functional tool that allows for variant calling and annotation of human mtDNA data coming from NGS experiments. The server was built with simplicity in mind, and builds on our own experience in interpreting mtDNA variants in the context of sudden death and rare diseases. Our objective is to provide an interface for non-bioinformaticians aiming to acquire (or contrast) mtDNA annotations via MToolBox. SG-ADVISER web server is freely available to all users at https://genomics.scripps.edu/mtdna .

[Efficient genome editing in human pluripotent stem cells through CRISPR/Cas9].

PubMed

Liu, Gai-gai; Li, Shuang; Wei, Yu-da; Zhang, Yong-xian; Ding, Qiu-rong

2015-11-01

The RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has offered a new platform for genome editing with high efficiency. Here, we report the use of CRISPR/Cas9 technology to target a specific genomic region in human pluripotent stem cells. We show that CRISPR/Cas9 can be used to disrupt a gene by introducing frameshift mutations to gene coding region; to knock in specific sequences (e.g. FLAG tag DNA sequence) to targeted genomic locus via homology directed repair; to induce large genomic deletion through dual-guide multiplex. Our results demonstrate the versatile application of CRISPR/Cas9 in stem cell genome editing, which can be widely utilized for functional studies of genes or genome loci in human pluripotent stem cells.

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response

PubMed Central

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications. PMID:25403473

RPA-coated single-stranded DNA as a platform for post-translational modifications in the DNA damage response.

PubMed

Maréchal, Alexandre; Zou, Lee

2015-01-01

The Replication Protein A (RPA) complex is an essential regulator of eukaryotic DNA metabolism. RPA avidly binds to single-stranded DNA (ssDNA) through multiple oligonucleotide/oligosaccharide-binding folds and coordinates the recruitment and exchange of genome maintenance factors to regulate DNA replication, recombination and repair. The RPA-ssDNA platform also constitutes a key physiological signal which activates the master ATR kinase to protect and repair stalled or collapsed replication forks during replication stress. In recent years, the RPA complex has emerged as a key target and an important regulator of post-translational modifications in response to DNA damage, which is critical for its genome guardian functions. Phosphorylation and SUMOylation of the RPA complex, and more recently RPA-regulated ubiquitination, have all been shown to control specific aspects of DNA damage signaling and repair by modulating the interactions between RPA and its partners. Here, we review our current understanding of the critical functions of the RPA-ssDNA platform in the maintenance of genome stability and its regulation through an elaborate network of covalent modifications.

Application of Quaternion in improving the quality of global sequence alignment scores for an ambiguous sequence target in Streptococcus pneumoniae DNA

NASA Astrophysics Data System (ADS)

Lestari, D.; Bustamam, A.; Novianti, T.; Ardaneswari, G.

2017-07-01

DNA sequence can be defined as a succession of letters, representing the order of nucleotides within DNA, using a permutation of four DNA base codes including adenine (A), guanine (G), cytosine (C), and thymine (T). The precise code of the sequences is determined using DNA sequencing methods and technologies, which have been developed since the 1970s and currently become highly developed, advanced and highly throughput sequencing technologies. So far, DNA sequencing has greatly accelerated biological and medical research and discovery. However, in some cases DNA sequencing could produce any ambiguous and not clear enough sequencing results that make them quite difficult to be determined whether these codes are A, T, G, or C. To solve these problems, in this study we can introduce other representation of DNA codes namely Quaternion Q = (PA, PT, PG, PC), where PA, PT, PG, PC are the probability of A, T, G, C bases that could appear in Q and PA + PT + PG + PC = 1. Furthermore, using Quaternion representations we are able to construct the improved scoring matrix for global sequence alignment processes, by applying a dot product method. Moreover, this scoring matrix produces better and higher quality of the match and mismatch score between two DNA base codes. In implementation, we applied the Needleman-Wunsch global sequence alignment algorithm using Octave, to analyze our target sequence which contains some ambiguous sequence data. The subject sequences are the DNA sequences of Streptococcus pneumoniae families obtained from the Genebank, meanwhile the target DNA sequence are received from our collaborator database. As the results we found the Quaternion representations improve the quality of the sequence alignment score and we can conclude that DNA sequence target has maximum similarity with Streptococcus pneumoniae.

High-coverage methylation data of a gene model before and after DNA damage and homologous repair.

PubMed

Pezone, Antonio; Russo, Giusi; Tramontano, Alfonso; Florio, Ermanno; Scala, Giovanni; Landi, Rosaria; Zuchegna, Candida; Romano, Antonella; Chiariotti, Lorenzo; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

2017-04-11

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.