Sample records for accurate bisulfite sequencing

  1. 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

  2. Hairpin Bisulfite Sequencing: Synchronous Methylation Analysis on Complementary DNA Strands of Individual Chromosomes.

    PubMed

    Giehr, Pascal; Walter, Jörn

    2018-01-01

    The accurate and quantitative detection of 5-methylcytosine is of great importance in the field of epigenetics. The method of choice is usually bisulfite sequencing because of the high resolution and the possibility to combine it with next generation sequencing. Nevertheless, also this method has its limitations. Following the bisulfite treatment DNA strands are no longer complementary such that in a subsequent PCR amplification the DNA methylation patterns information of only one of the two DNA strand is preserved. Several years ago Hairpin Bisulfite sequencing was developed as a method to obtain the pattern information on complementary DNA strands. The method requires fragmentation (usually by enzymatic cleavage) of genomic DNA followed by a covalent linking of both DNA strands through ligation of a short DNA hairpin oligonucleotide to both strands. The ligated covalently linked dsDNA products are then subjected to a conventional bisulfite treatment during which all unmodified cytosines are converted to uracils. During the treatment the DNA is denatured forming noncomplementary ssDNA circles. These circles serve as a template for a locus specific PCR to amplify chromosomal patterns of the region of interest. As a result one ends up with a linearized product, which contains the methylation information of both complementary DNA strands.

  3. 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.

  4. Partial bisulfite conversion for unique template sequencing

    PubMed Central

    Kumar, Vijay; Rosenbaum, Julie; Wang, Zihua; Forcier, Talitha; Ronemus, Michael; Wigler, Michael

    2018-01-01

    Abstract We introduce a new protocol, mutational sequencing or muSeq, which uses sodium bisulfite to randomly deaminate unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling 135 000 restriction fragments in a PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone. PMID:29161423

  5. MethylViewer: computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects.

    PubMed

    Pardo, Carolina E; Carr, Ian M; Hoffman, Christopher J; Darst, Russell P; Markham, Alexander F; Bonthron, David T; Kladde, Michael P

    2011-01-01

    Bisulfite sequencing is a widely-used technique for examining cytosine DNA methylation at nucleotide resolution along single DNA strands. Probing with cytosine DNA methyltransferases followed by bisulfite sequencing (MAPit) is an effective technique for mapping protein-DNA interactions. Here, MAPit methylation footprinting with M.CviPI, a GC methyltransferase we previously cloned and characterized, was used to probe hMLH1 chromatin in HCT116 and RKO colorectal cancer cells. Because M.CviPI-probed samples contain both CG and GC methylation, we developed a versatile, visually-intuitive program, called MethylViewer, for evaluating the bisulfite sequencing results. Uniquely, MethylViewer can simultaneously query cytosine methylation status in bisulfite-converted sequences at as many as four different user-defined motifs, e.g. CG, GC, etc., including motifs with degenerate bases. Data can also be exported for statistical analysis and as publication-quality images. Analysis of hMLH1 MAPit data with MethylViewer showed that endogenous CG methylation and accessible GC sites were both mapped on single molecules at high resolution. Disruption of positioned nucleosomes on single molecules of the PHO5 promoter was detected in budding yeast using M.CviPII, increasing the number of enzymes available for probing protein-DNA interactions. MethylViewer provides an integrated solution for primer design and rapid, accurate and detailed analysis of bisulfite sequencing or MAPit datasets from virtually any biological or biochemical system.

  6. Partial bisulfite conversion for unique template sequencing.

    PubMed

    Kumar, Vijay; Rosenbaum, Julie; Wang, Zihua; Forcier, Talitha; Ronemus, Michael; Wigler, Michael; Levy, Dan

    2018-01-25

    We introduce a new protocol, mutational sequencing or muSeq, which uses sodium bisulfite to randomly deaminate unmethylated cytosines at a fixed and tunable rate. The muSeq protocol marks each initial template molecule with a unique mutation signature that is present in every copy of the template, and in every fragmented copy of a copy. In the sequenced read data, this signature is observed as a unique pattern of C-to-T or G-to-A nucleotide conversions. Clustering reads with the same conversion pattern enables accurate count and long-range assembly of initial template molecules from short-read sequence data. We explore count and low-error sequencing by profiling 135 000 restriction fragments in a PstI representation, demonstrating that muSeq improves copy number inference and significantly reduces sporadic sequencer error. We explore long-range assembly in the context of cDNA, generating contiguous transcript clusters greater than 3,000 bp in length. The muSeq assemblies reveal transcriptional diversity not observable from short-read data alone. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Comparison and quantitative verification of mapping algorithms for whole genome bisulfite sequencing

    USDA-ARS?s Scientific Manuscript database

    Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitat...

  8. Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.

    PubMed

    Li, Qing; Hermanson, Peter J; Springer, Nathan M

    2018-01-01

    DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. Coupled with next-generation sequencing, whole-genome bisulfite sequencing (WGBS) allows DNA methylation to be evaluated at a genome-wide scale. Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.

  9. 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

  10. BS-virus-finder: virus integration calling using bisulfite sequencing data.

    PubMed

    Gao, Shengjie; Hu, Xuesong; Xu, Fengping; Gao, Changduo; Xiong, Kai; Zhao, Xiao; Chen, Haixiao; Zhao, Shancen; Wang, Mengyao; Fu, Dongke; Zhao, Xiaohui; Bai, Jie; Mao, Likai; Li, Bo; Wu, Song; Wang, Jian; Li, Shengbin; Yang, Huangming; Bolund, Lars; Pedersen, Christian N S

    2018-01-01

    DNA methylation plays a key role in the regulation of gene expression and carcinogenesis. Bisulfite sequencing studies mainly focus on calling single nucleotide polymorphism, different methylation region, and find allele-specific DNA methylation. Until now, only a few software tools have focused on virus integration using bisulfite sequencing data. We have developed a new and easy-to-use software tool, named BS-virus-finder (BSVF, RRID:SCR_015727), to detect viral integration breakpoints in whole human genomes. The tool is hosted at https://github.com/BGI-SZ/BSVF. BS-virus-finder demonstrates high sensitivity and specificity. It is useful in epigenetic studies and to reveal the relationship between viral integration and DNA methylation. BS-virus-finder is the first software tool to detect virus integration loci by using bisulfite sequencing data. © The Authors 2017. Published by Oxford University Press.

  11. BiQ Analyzer HT: locus-specific analysis of DNA methylation by high-throughput bisulfite sequencing

    PubMed Central

    Lutsik, Pavlo; Feuerbach, Lars; Arand, Julia; Lengauer, Thomas; Walter, Jörn; Bock, Christoph

    2011-01-01

    Bisulfite sequencing is a widely used method for measuring DNA methylation in eukaryotic genomes. The assay provides single-base pair resolution and, given sufficient sequencing depth, its quantitative accuracy is excellent. High-throughput sequencing of bisulfite-converted DNA can be applied either genome wide or targeted to a defined set of genomic loci (e.g. using locus-specific PCR primers or DNA capture probes). Here, we describe BiQ Analyzer HT (http://biq-analyzer-ht.bioinf.mpi-inf.mpg.de/), a user-friendly software tool that supports locus-specific analysis and visualization of high-throughput bisulfite sequencing data. The software facilitates the shift from time-consuming clonal bisulfite sequencing to the more quantitative and cost-efficient use of high-throughput sequencing for studying locus-specific DNA methylation patterns. In addition, it is useful for locus-specific visualization of genome-wide bisulfite sequencing data. PMID:21565797

  12. Evaluation of Different Oligonucleotide Base Substitutions at CpG Binding sites in Multiplex Bisulfite-PCR sequencing.

    PubMed

    Lu, Jennifer; Ru, Kelin; Candiloro, Ida; Dobrovic, Alexander; Korbie, Darren; Trau, Matt

    2017-03-22

    Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG's contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed- or deoxyinosine- base modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates.

  13. Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

    PubMed

    Olova, Nelly; Krueger, Felix; Andrews, Simon; Oxley, David; Berrens, Rebecca V; Branco, Miguel R; Reik, Wolf

    2018-03-15

    Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

  14. Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection.

    PubMed

    Owa, Chie; Poulin, Matthew; Yan, Liying; Shioda, Toshi

    2018-01-01

    The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis

  15. Detection of Cytosine methylation in ancient DNA from five native american populations using bisulfite sequencing.

    PubMed

    Smith, Rick W A; Monroe, Cara; Bolnick, Deborah A

    2015-01-01

    While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.

  16. Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA.

    PubMed

    Dikow, Nicola; Nygren, Anders Oh; Schouten, Jan P; Hartmann, Carolin; Krämer, Nikola; Janssen, Bart; Zschocke, Johannes

    2007-06-01

    Standard methods used for genomic methylation analysis allow the detection of complete absence of either methylated or non-methylated alleles but are usually unable to detect changes in the proportion of methylated and unmethylated alleles. We compare two methods for quantitative methylation analysis, using the chromosome 15q11-q13 imprinted region as model. Absence of the non-methylated paternal allele in this region leads to Prader-Willi syndrome (PWS) whilst absence of the methylated maternal allele results in Angelman syndrome (AS). A proportion of AS is caused by mosaic imprinting defects which may be missed with standard methods and require quantitative analysis for their detection. Sequence-based quantitative methylation analysis (SeQMA) involves quantitative comparison of peaks generated through sequencing reactions after bisulfite treatment. It is simple, cost-effective and can be easily established for a large number of genes. However, our results support previous suggestions that methods based on bisulfite treatment may be problematic for exact quantification of methylation status. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) avoids bisulfite treatment. It detects changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in one simple reaction. Once established in a laboratory setting, the method is more accurate, reliable and less time consuming.

  17. Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing.

    PubMed

    Guo, Hongshan; Zhu, Ping; Guo, Fan; Li, Xianlong; Wu, Xinglong; Fan, Xiaoying; Wen, Lu; Tang, Fuchou

    2015-05-01

    The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution. Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified the original RRBS method by integrating all the experimental steps before PCR amplification into a single-tube reaction. These modifications enable scRRBS to provide digitized methylation information on ∼1 million CpG sites within an individual diploid mouse or human cell at single-base resolution. Compared with the single-cell bisulfite sequencing (scBS) technique, scRRBS covers fewer CpG sites, but it provides better coverage for CpG islands (CGIs), which are likely to be the most informative elements for DNA methylation. The entire procedure takes ∼3 weeks, and it requires strong molecular biology skills.

  18. 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.

  19. 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

  20. A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms.

    PubMed

    Leakey, Tatiana I; Zielinski, Jerzy; Siegfried, Rachel N; Siegel, Eric R; Fan, Chun-Yang; Cooney, Craig A

    2008-06-01

    DNA methylation at cytosines is a widely studied epigenetic modification. Methylation is commonly detected using bisulfite modification of DNA followed by PCR and additional techniques such as restriction digestion or sequencing. These additional techniques are either laborious, require specialized equipment, or are not quantitative. Here we describe a simple algorithm that yields quantitative results from analysis of conventional four-dye-trace sequencing. We call this method Mquant and we compare it with the established laboratory method of combined bisulfite restriction assay (COBRA). This analysis of sequencing electropherograms provides a simple, easily applied method to quantify DNA methylation at specific CpG sites.

  1. Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions.

    PubMed

    Schmidt, Martin; Van Bel, Michiel; Woloszynska, Magdalena; Slabbinck, Bram; Martens, Cindy; De Block, Marc; Coppens, Frederik; Van Lijsebettens, Mieke

    2017-07-06

    Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant

  2. Direct bisulfite sequencing for examination of DNA methylation with gene and nucleotide resolution from brain tissues.

    PubMed

    Parrish, R Ryley; Day, Jeremy J; Lubin, Farah D

    2012-07-01

    DNA methylation is an epigenetic modification that is essential for the development and mature function of the central nervous system. Due to the relevance of this modification to the transcriptional control of gene expression, it is often necessary to examine changes in DNA methylation patterns with both gene and single-nucleotide resolution. Here, we describe an in-depth basic protocol for direct bisulfite sequencing of DNA isolated from brain tissue, which will permit direct assessment of methylation status at individual genes as well as individual cytosine molecules/nucleotides within a genomic region. This method yields analysis of DNA methylation patterns that is robust, accurate, and reproducible, thereby allowing insights into the role of alterations in DNA methylation in brain tissue.

  3. Maximizing ecological and evolutionary insight in bisulfite sequencing data sets

    PubMed Central

    Lea, Amanda J.; Vilgalys, Tauras P.; Durst, Paul A.P.; Tung, Jenny

    2017-01-01

    Preface Genome-scale bisulfite sequencing approaches have opened the door to ecological and evolutionary studies of DNA methylation in many organisms. These approaches can be powerful. However, they introduce new methodological and statistical considerations, some of which are particularly relevant to non-model systems. Here, we highlight how these considerations influence a study’s power to link methylation variation with a predictor variable of interest. Relative to current practice, we argue that sample sizes will need to increase to provide robust insights. We also provide recommendations for overcoming common challenges and an R Shiny app to aid in study design. PMID:29046582

  4. Detecting differential DNA methylation from sequencing of bisulfite converted DNA of diverse species.

    PubMed

    Huh, Iksoo; Wu, Xin; Park, Taesung; Yi, Soojin V

    2017-07-21

    DNA methylation is one of the most extensively studied epigenetic modifications of genomic DNA. In recent years, sequencing of bisulfite-converted DNA, particularly via next-generation sequencing technologies, has become a widely popular method to study DNA methylation. This method can be readily applied to a variety of species, dramatically expanding the scope of DNA methylation studies beyond the traditionally studied human and mouse systems. In parallel to the increasing wealth of genomic methylation profiles, many statistical tools have been developed to detect differentially methylated loci (DMLs) or differentially methylated regions (DMRs) between biological conditions. We discuss and summarize several key properties of currently available tools to detect DMLs and DMRs from sequencing of bisulfite-converted DNA. However, the majority of the statistical tools developed for DML/DMR analyses have been validated using only mammalian data sets, and less priority has been placed on the analyses of invertebrate or plant DNA methylation data. We demonstrate that genomic methylation profiles of non-mammalian species are often highly distinct from those of mammalian species using examples of honey bees and humans. We then discuss how such differences in data properties may affect statistical analyses. Based on these differences, we provide three specific recommendations to improve the power and accuracy of DML and DMR analyses of invertebrate data when using currently available statistical tools. These considerations should facilitate systematic and robust analyses of DNA methylation from diverse species, thus advancing our understanding of DNA methylation. © The Author 2017. Published by Oxford University Press.

  5. Genome-wide bisulfite sensitivity profiling of yeast suggests bisulfite inhibits transcription.

    PubMed

    Segovia, Romulo; Mathew, Veena; Tam, Annie S; Stirling, Peter C

    2017-09-01

    Bisulfite, in the form of sodium bisulfite or metabisulfite, is used commercially as a food preservative. Bisulfite is used in the laboratory as a single-stranded DNA mutagen in epigenomic analyses of DNA methylation. Recently it has also been used on whole yeast cells to induce mutations in exposed single-stranded regions in vivo. To understand the effects of bisulfite on live cells we conducted a genome-wide screen for bisulfite sensitive mutants in yeast. Screening the deletion mutant array, and collections of essential gene mutants we define a genetic network of bisulfite sensitive mutants. Validation of screen hits revealed hyper-sensitivity of transcription and RNA processing mutants, rather than DNA repair pathways and follow-up analyses support a role in perturbation of RNA transactions. We propose a model in which bisulfite-modified nucleotides may interfere with transcription or RNA metabolism when used in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. GPU-BSM: A GPU-Based Tool to Map Bisulfite-Treated Reads

    PubMed Central

    Manconi, Andrea; Orro, Alessandro; Manca, Emanuele; Armano, Giuliano; Milanesi, Luciano

    2014-01-01

    Cytosine DNA methylation is an epigenetic mark implicated in several biological processes. Bisulfite treatment of DNA is acknowledged as the gold standard technique to study methylation. This technique introduces changes in the genomic DNA by converting cytosines to uracils while 5-methylcytosines remain nonreactive. During PCR amplification 5-methylcytosines are amplified as cytosine, whereas uracils and thymines as thymine. To detect the methylation levels, reads treated with the bisulfite must be aligned against a reference genome. Mapping these reads to a reference genome represents a significant computational challenge mainly due to the increased search space and the loss of information introduced by the treatment. To deal with this computational challenge we devised GPU-BSM, a tool based on modern Graphics Processing Units. Graphics Processing Units are hardware accelerators that are increasingly being used successfully to accelerate general-purpose scientific applications. GPU-BSM is a tool able to map bisulfite-treated reads from whole genome bisulfite sequencing and reduced representation bisulfite sequencing, and to estimate methylation levels, with the goal of detecting methylation. Due to the massive parallelization obtained by exploiting graphics cards, GPU-BSM aligns bisulfite-treated reads faster than other cutting-edge solutions, while outperforming most of them in terms of unique mapped reads. PMID:24842718

  7. Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR

    PubMed Central

    Miner, Brooks E.; Stöger, Reinhard J.; Burden, Alice F.; Laird, Charles D.; Hansen, R. Scott

    2004-01-01

    PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular ‘batch-stamps’ that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes. PMID:15459281

  8. Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

    PubMed Central

    Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

    2013-01-01

    Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

  9. Methylation analysis of plasma cell-free DNA for breast cancer early detection using bisulfite next-generation sequencing.

    PubMed

    Li, Zibo; Guo, Xinwu; Tang, Lili; Peng, Limin; Chen, Ming; Luo, Xipeng; Wang, Shouman; Xiao, Zhi; Deng, Zhongping; Dai, Lizhong; Xia, Kun; Wang, Jun

    2016-10-01

    Circulating cell-free DNA (cfDNA) has been considered as a potential biomarker for non-invasive cancer detection. To evaluate the methylation levels of six candidate genes (EGFR, GREM1, PDGFRB, PPM1E, SOX17, and WRN) in plasma cfDNA as biomarkers for breast cancer early detection, quantitative analysis of the promoter methylation of these genes from 86 breast cancer patients and 67 healthy controls was performed by using microfluidic-PCR-based target enrichment and next-generation bisulfite sequencing technology. The predictive performance of different logistic models based on methylation status of candidate genes was investigated by means of the area under the ROC curve (AUC) and odds ratio (OR) analysis. Results revealed that EGFR, PPM1E, and 8 gene-specific CpG sites showed significantly hypermethylation in cancer patients' plasma and significantly associated with breast cancer (OR ranging from 2.51 to 9.88). The AUC values for these biomarkers were ranging from 0.66 to 0.75. Combinations of multiple hypermethylated genes or CpG sites substantially improved the predictive performance for breast cancer detection. Our study demonstrated the feasibility of quantitative measurement of candidate gene methylation in cfDNA by using microfluidic-PCR-based target enrichment and bisulfite next-generation sequencing, which is worthy of further validation and potentially benefits a broad range of applications in clinical oncology practice. Quantitative analysis of methylation pattern of plasma cfDNA by next-generation sequencing might be a valuable non-invasive tool for early detection of breast cancer.

  10. 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 .

  11. Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

    PubMed

    Clark, Stephen J; Smallwood, Sébastien A; Lee, Heather J; Krueger, Felix; Reik, Wolf; Kelsey, Gavin

    2017-03-01

    DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

  12. Pipeline for large-scale microdroplet bisulfite PCR-based sequencing allows the tracking of hepitype evolution in tumors.

    PubMed

    Herrmann, Alexander; Haake, Andrea; Ammerpohl, Ole; Martin-Guerrero, Idoia; Szafranski, Karol; Stemshorn, Kathryn; Nothnagel, Michael; Kotsopoulos, Steve K; Richter, Julia; Warner, Jason; Olson, Jeff; Link, Darren R; Schreiber, Stefan; Krawczak, Michael; Platzer, Matthias; Nürnberg, Peter; Siebert, Reiner; Hampe, Jochen

    2011-01-01

    Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into "hepitypes" and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer.

  13. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium bisulfite. 182.3616 Section 182.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations...

  14. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium bisulfite. 182.3616 Section 182.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations...

  15. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations...

  16. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium bisulfite. 182.3616 Section 182.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations...

  17. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations...

  18. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations...

  19. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium bisulfite. 182.3616 Section 182.3616...) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Chemical Preservatives § 182.3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or explanation. This substance is...

  20. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations...

  1. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations...

  2. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium bisulfite. 182.3616 Section 182.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  3. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Sodium bisulfite. (a) Product. Sodium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  4. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Sodium bisulfite. (a) Product. Sodium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  5. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Sodium bisulfite. (a) Product. Sodium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  6. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  7. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Sodium bisulfite. (a) Product. Sodium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  8. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  9. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  10. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  11. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  12. Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing.

    PubMed

    Leontiou, Chrysanthia A; Hadjidaniel, Michael D; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C

    2015-01-01

    Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite

  13. Performances of Different Fragment Sizes for Reduced Representation Bisulfite Sequencing in Pigs.

    PubMed

    Yuan, Xiao-Long; Zhang, Zhe; Pan, Rong-Yang; Gao, Ning; Deng, Xi; Li, Bin; Zhang, Hao; Sangild, Per Torp; Li, Jia-Qi

    2017-01-01

    Reduced representation bisulfite sequencing (RRBS) has been widely used to profile genome-scale DNA methylation in mammalian genomes. However, the applications and technical performances of RRBS with different fragment sizes have not been systematically reported in pigs, which serve as one of the important biomedical models for humans. The aims of this study were to evaluate capacities of RRBS libraries with different fragment sizes to characterize the porcine genome. We found that the Msp I-digested segments between 40 and 220 bp harbored a high distribution peak at 74 bp, which were highly overlapped with the repetitive elements and might reduce the unique mapping alignment. The RRBS library of 110-220 bp fragment size had the highest unique mapping alignment and the lowest multiple alignment. The cost-effectiveness of the 40-110 bp, 110-220 bp and 40-220 bp fragment sizes might decrease when the dataset size was more than 70, 50 and 110 million reads for these three fragment sizes, respectively. Given a 50-million dataset size, the average sequencing depth of the detected CpG sites in the 110-220 bp fragment size appeared to be deeper than in the 40-110 bp and 40-220 bp fragment sizes, and these detected CpG sties differently located in gene- and CpG island-related regions. In this study, our results demonstrated that selections of fragment sizes could affect the numbers and sequencing depth of detected CpG sites as well as the cost-efficiency. No single solution of RRBS is optimal in all circumstances for investigating genome-scale DNA methylation. This work provides the useful knowledge on designing and executing RRBS for investigating the genome-wide DNA methylation in tissues from pigs.

  14. Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

    PubMed Central

    Su, Chang; Wang, Chao; He, Lin; Yang, Chuanping; Wang, Yucheng

    2014-01-01

    DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch) by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees. PMID:25514241

  15. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Chemical Preservatives § 182.3739 Sodium bisulfite. (a) Product. Sodium...

  16. Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

    PubMed Central

    Leontiou, Chrysanthia A.; Hadjidaniel, Michael D.; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C.

    2015-01-01

    Introduction Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Methods Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. Results The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Conclusion Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed

  17. Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing.

    PubMed

    Yuan, Xiao-Long; Gao, Ning; Xing, Yan; Zhang, Hai-Bin; Zhang, Ai-Ling; Liu, Jing; He, Jin-Long; Xu, Yuan; Lin, Wen-Mian; Chen, Zan-Mou; Zhang, Hao; Zhang, Zhe; Li, Jia-Qi

    2016-02-25

    Substantial evidence has shown that DNA methylation regulates the initiation of ovarian and sexual maturation. Here, we investigated the genome-wide profile of DNA methylation in porcine ovaries at single-base resolution using reduced representation bisulfite sequencing. The biological variation was minimal among the three ovarian replicates. We found hypermethylation frequently occurred in regions with low gene abundance, while hypomethylation in regions with high gene abundance. The DNA methylation around transcriptional start sites was negatively correlated with their own CpG content. Additionally, the methylation level in the bodies of genes was higher than that in their 5' and 3' flanking regions. The DNA methylation pattern of the low CpG content promoter genes differed obviously from that of the high CpG content promoter genes. The DNA methylation level of the porcine ovary was higher than that of the porcine intestine. Analyses of the genome-wide DNA methylation in porcine ovaries would advance the knowledge and understanding of the porcine ovarian methylome.

  18. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.620 Menadione dimethylpyrimidinol bisulfite. The food additive... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620...

  19. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.620 Menadione dimethylpyrimidinol bisulfite. The food additive... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620...

  20. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.620 Menadione dimethylpyrimidinol bisulfite. The food additive... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620...

  1. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.620 Menadione dimethylpyrimidinol bisulfite. The food additive... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620...

  2. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.620 Menadione dimethylpyrimidinol bisulfite. The food additive... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620...

  3. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  4. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  5. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  6. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  7. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  8. Final report on the safety assessment of sodium sulfite, potassium sulfite, ammonium sulfite, sodium bisulfite, ammonium bisulfite, sodium metabisulfite and potassium metabisulfite.

    PubMed

    Nair, Bindu; Elmore, Amy R

    2003-01-01

    Sodium Sulfite, Ammonium Sulfite, Sodium Bisulfite, Potassium Bisulfite, Ammonium Bisulfite, Sodium Metabisulfite, and Potassium Metabisulfite are inorganic salts that function as reducing agents in cosmetic formulations. All except Sodium Metabisulfite also function as hair-waving/straightening agents. In addition, Sodium Sulfite, Potassium Sulfite, Sodium Bisulfite, and Sodium Metabisulfite function as antioxidants. Although Ammonium Sulfite is not in current use, the others are widely used in hair care products. Sulfites that enter mammals via ingestion, inhalation, or injection are metabolized by sulfite oxidase to sulfate. In oral-dose animal toxicity studies, hyperplastic changes in the gastric mucosa were the most common findings at high doses. Ammonium Sulfite aerosol had an acute LC(50) of >400 mg/m(3) in guinea pigs. A single exposure to low concentrations of a Sodium Sulfite fine aerosol produced dose-related changes in the lung capacity parameters of guinea pigs. A 3-day exposure of rats to a Sodium Sulfite fine aerosol produced mild pulmonary edema and irritation of the tracheal epithelium. Severe epithelial changes were observed in dogs exposed for 290 days to 1 mg/m(3) of a Sodium Metabisulfite fine aerosol. These fine aerosols contained fine respirable particle sizes that are not found in cosmetic aerosols or pump sprays. None of the cosmetic product types, however, in which these ingredients are used are aerosolized. Sodium Bisulfite (tested at 38%) and Sodium Metabisulfite (undiluted) were not irritants to rabbits following occlusive exposures. Sodium Metabisulfite (tested at 50%) was irritating to guinea pigs following repeated exposure. In rats, Sodium Sulfite heptahydrate at large doses (up to 3.3 g/kg) produced fetal toxicity but not teratogenicity. Sodium Bisulfite, Sodium Metabisulfite, and Potassium Metabisulfite were not teratogenic for mice, rats, hamsters, or rabbits at doses up to 160 mg/kg. Generally, Sodium Sulfite, Sodium

  9. An information-theoretic approach to the modeling and analysis of whole-genome bisulfite sequencing data.

    PubMed

    Jenkinson, Garrett; Abante, Jordi; Feinberg, Andrew P; Goutsias, John

    2018-03-07

    DNA methylation is a stable form of epigenetic memory used by cells to control gene expression. Whole genome bisulfite sequencing (WGBS) has emerged as a gold-standard experimental technique for studying DNA methylation by producing high resolution genome-wide methylation profiles. Statistical modeling and analysis is employed to computationally extract and quantify information from these profiles in an effort to identify regions of the genome that demonstrate crucial or aberrant epigenetic behavior. However, the performance of most currently available methods for methylation analysis is hampered by their inability to directly account for statistical dependencies between neighboring methylation sites, thus ignoring significant information available in WGBS reads. We present a powerful information-theoretic approach for genome-wide modeling and analysis of WGBS data based on the 1D Ising model of statistical physics. This approach takes into account correlations in methylation by utilizing a joint probability model that encapsulates all information available in WGBS methylation reads and produces accurate results even when applied on single WGBS samples with low coverage. Using the Shannon entropy, our approach provides a rigorous quantification of methylation stochasticity in individual WGBS samples genome-wide. Furthermore, it utilizes the Jensen-Shannon distance to evaluate differences in methylation distributions between a test and a reference sample. Differential performance assessment using simulated and real human lung normal/cancer data demonstrate a clear superiority of our approach over DSS, a recently proposed method for WGBS data analysis. Critically, these results demonstrate that marginal methods become statistically invalid when correlations are present in the data. This contribution demonstrates clear benefits and the necessity of modeling joint probability distributions of methylation using the 1D Ising model of statistical physics and of

  10. Detoxification of Dissolved SO2 (Bisulfite) by Terricolous Mosses

    PubMed Central

    BHARALI, BHAGAWAN; BATES, JEFFREY W.

    2006-01-01

    • Background and Aims The widespread calcifuge moss Pleurozium schreberi is moderately tolerant of SO2, whereas Rhytidiadelphus triquetrus is limited to calcareous soils in regions of the UK that were strongly affected by SO2 pollution in the 20th century. The proposition that tolerance of SO2 by these terricolous mosses depends on metabolic detoxification of dissolved bisulfite was investigated. • Methods The capacities of the two mosses to accelerate loss of bisulfite from aqueous solutions of NaHSO3 were studied using DTNB [5, 5-dithio-(2-nitrobenzoic acid)] to assay bisulfite, and HPLC to assay sulfate in the incubation solutions. Incubations were performed for different durations, in the presence and absence of light, at a range of solution pH values, in the presence of metabolic inhibitors and with altered moss apoplastic Ca2+ and Fe3+ levels. • Key Results Bisulfite disappearance was markedly stimulated in the light and twice as great for R. triquetrus as for P. schreberi. DCMU, an inhibitor of photosynthetic electron chain transport, significantly reduced bisufite loss. • Conclusions Bisulfite (SO2) tolerance in these terricolous mosses involves extracellular oxidation using metabolic (photo-oxidative) energy, passive oxidation by adsorbed Fe3+ (only available to the calcifuge) and probably also internal metabolic detoxification. PMID:16319108

  11. Base-resolution detection of N 4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite-sequencing

    DOE PAGES

    Yu, Miao; Ji, Lexiang; Neumann, Drexel A.; ...

    2015-07-15

    Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N 6-methyladenine (6mA), 5-methylcytosine (5mC) and N 4-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method thatmore » rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. Lastly, in combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.« less

  12. Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

    PubMed

    Maggi, Elaine C; Gravina, Silvia; Cheng, Haiying; Piperdi, Bilal; Yuan, Ziqiang; Dong, Xiao; Libutti, Steven K; Vijg, Jan; Montagna, Cristina

    2018-01-01

    The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

  13. Identification of Smoking-Associated Differentially Methylated Regions Using Reduced Representation Bisulfite Sequencing and Cell type-Specific Enhancer Activation and Gene Expression.

    PubMed

    Wan, Ma; Bennett, Brian D; Pittman, Gary S; Campbell, Michelle R; Reynolds, Lindsay M; Porter, Devin K; Crowl, Christopher L; Wang, Xuting; Su, Dan; Englert, Neal A; Thompson, Isabel J; Liu, Yongmei; Bell, Douglas A

    2018-04-27

    Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure. Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes. We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating CD14+ monocyte DNA collected from two independent human studies [ n =38 from Clinical Research Unit (CRU) and n =55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4+ T cells, CD8+ T cells, CD15+ granulocytes, CD19+ B cells, and CD56+ NK cells ( n =19 females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA. RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene ( AHRR ) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR , C5orf55-EXOC-AS , and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers. Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type-specific activation

  14. Genome-wide oxidative bisulfite sequencing identifies sex-specific methylation differences in the human placenta

    PubMed Central

    Johnson, Michelle D; Dopierala, Justyna

    2018-01-01

    ABSTRACT DNA methylation is an important regulator of gene function. Fetal sex is associated with the risk of several specific pregnancy complications related to placental function. However, the association between fetal sex and placental DNA methylation remains poorly understood. We carried out whole-genome oxidative bisulfite sequencing in the placentas of two healthy female and two healthy male pregnancies generating an average genome depth of coverage of 25x. Most highly ranked differentially methylated regions (DMRs) were located on the X chromosome but we identified a 225 kb sex-specific DMR in the body of the CUB and Sushi Multiple Domains 1 (CSMD1) gene on chromosome 8. The sex-specific differential methylation pattern observed in this region was validated in additional placentas using in-solution target capture. In a new RNA-seq data set from 64 female and 67 male placentas, CSMD1 mRNA was 1.8-fold higher in male than in female placentas (P value = 8.5 × 10−7, Mann-Whitney test). Exon-level quantification of CSMD1 mRNA from these 131 placentas suggested a likely placenta-specific CSMD1 isoform not detected in the 21 somatic tissues analyzed. We show that the gene body of an autosomal gene, CSMD1, is differentially methylated in a sex- and placental-specific manner, displaying sex-specific differences in placental transcript abundance. PMID:29376485

  15. Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

    USDA-ARS?s Scientific Manuscript database

    Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylat...

  16. An optimized rapid bisulfite conversion method with high recovery of cell-free DNA.

    PubMed

    Yi, Shaohua; Long, Fei; Cheng, Juanbo; Huang, Daixin

    2017-12-19

    Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA. We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods. The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

  17. A colorimetric and fluorogenic probe for bisulfite using benzopyrylium as the recognition unit.

    PubMed

    Zhang, Yun; Zhang, Xiangwen; Yang, Xiao-Feng; Zhang, Juan

    2017-11-01

    A coumarin-benzopyrylium (CB) platform has been developed for the colorimetric and fluorogenic detection of bisulfite. The proposed probe utilizes coumarin as the fluorophore and positively charged benzopyrylium as the reaction site. The method employs the nucleophilic addition of bisulfite to the benzopyrylium moiety of CB to inactivate the electron-deficient oxonium ion. The driving force for photo-induced electron transfer is considerably diminished, thereby promoting the emission intensity of the coumarin fluorophore. The fluorescence intensity at 510 nm is linear with bisulfite concentration over a range of 0.2-7.5 μM with a detection limit of 42 nM (3δ). CB shows a rapid response (within 30 s) and high selectivity and sensitivity for bisulfite. Preliminary studies show that CB has great potential for bisulfite detection in real samples and in living cells. Copyright © 2017 John Wiley & Sons, Ltd.

  18. Existence of the sugar-bisulfite adducts and its inhibiting effect on degradation of monosaccharide in acid system.

    PubMed

    Shi, Yan

    2014-02-01

    Degradation of fermentable monosaccharides is one of the primary concerns for acid prehydrolysis of lignocellulosic biomass. Recently, in our research on degradation of pure monosaccharides in aqueous SO₂ solution by gas chromatography (GC) analysis, we found that detected yield was not actual yield of each monosaccharide due to the existence of sugar-bisulfite adducts, and a new method was developed by ourselves which led to accurate detection of recovery yield of each monosaccharide in aqueous SO₂ solution by GC analysis. By the use of this method, degradation of each monosaccharide in aqueous SO₂ was investigated and results showed that sugar-bisulfite adducts have different inhibiting effect on degradation of each monosaccharide in aqueous SO₂ because of their different stability. In addition, NMR testing also demonstrated possible existence of reaction between conjugated based HSO₃(-) and aldehyde group of sugars in acid system.

  19. Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

    PubMed

    Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M; Michor, Franziska; Fan, Rong; Pan, Xinghua

    2017-06-02

    Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

    PubMed Central

    Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L.; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M.

    2017-01-01

    Abstract Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. PMID:28126923

  1. CpG location and methylation level are crucial factors for the early detection of oral squamous cell carcinoma in brushing samples using bisulfite sequencing of a 13-gene panel.

    PubMed

    Morandi, Luca; Gissi, Davide; Tarsitano, Achille; Asioli, Sofia; Gabusi, Andrea; Marchetti, Claudio; Montebugnoli, Lucio; Foschini, Maria Pia

    2017-01-01

    Oral squamous cell carcinoma (OSCC) is usually diagnosed at an advanced stage and is commonly preceded by oral premalignant lesions. The mortality rates have remained unchanged (50% within 5 years after diagnosis), and it is related to tobacco smoking and alcohol intake. Novel molecular markers for early diagnosis are urgently needed. The purpose of this study was to evaluate the diagnostic value of methylation level in a set of 18 genes by bisulfite next-generation sequencing. With minimally invasive oral brushing, 28 consecutive OSCC, one squamous cell carcinoma with sarcomatoid features, six high-grade squamous intraepithelial lesions (HGSIL), 30 normal contralateral mucosa from the same patients, and 65 healthy donors were evaluated for DNA methylation analyzing 18 target genes by quantitative bisulfite next-generation sequencing. We further evaluated an independent cohort (validation dataset) made of 20 normal donors, one oral fibroma, 14 oral lichen planus (OLP), three proliferative verrucous leukoplakia (PVL), and two OSCC. Comparing OSCC with normal healthy donors and contralateral mucosa in 355 CpGs, we identified the following epigenetically altered genes: ZAP70 , ITGA4 , KIF1A , PARP15 , EPHX3 , NTM , LRRTM1 , FLI1 , MIR193 , LINC00599 , PAX1 , and MIR137HG showing hypermethylation and MIR296 , TERT , and GP1BB showing hypomethylation . The behavior of ZAP70 , GP1BB , H19 , EPHX3 , and MIR193 fluctuated among different interrogated CpGs. The gap between normal and OSCC samples remained mostly the same (Kruskal-Wallis P values < 0.05), but the absolute values changed conspicuously. ROC curve analysis identified the most informative CpGs, and we correctly stratified OSCC and HGSIL from normal donors using a multiclass linear discriminant analysis in a 13-gene panel (AUC 0.981). Only the OSCC with sarcomatoid features was negative. Three contralateral mucosa were positive, a sign of a possible field cancerization. Among imprinted genes, only MIR296

  2. PrimerSuite: A High-Throughput Web-Based Primer Design Program for Multiplex Bisulfite PCR.

    PubMed

    Lu, Jennifer; Johnston, Andrew; Berichon, Philippe; Ru, Ke-Lin; Korbie, Darren; Trau, Matt

    2017-01-24

    The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously (PrimerSuite), (ii) screen for primer-primer dimerizing artefacts (PrimerDimer), and (iii) support multiplex PCR assays (PrimerPlex). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com).

  3. Arbitrarily accurate twin composite π -pulse sequences

    NASA Astrophysics Data System (ADS)

    Torosov, Boyan T.; Vitanov, Nikolay V.

    2018-04-01

    We present three classes of symmetric broadband composite pulse sequences. The composite phases are given by analytic formulas (rational fractions of π ) valid for any number of constituent pulses. The transition probability is expressed by simple analytic formulas and the order of pulse area error compensation grows linearly with the number of pulses. Therefore, any desired compensation order can be produced by an appropriate composite sequence; in this sense, they are arbitrarily accurate. These composite pulses perform equally well as or better than previously published ones. Moreover, the current sequences are more flexible as they allow total pulse areas of arbitrary integer multiples of π .

  4. Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing.

    PubMed

    Crampton, Mollee; Sripathi, Venkateswara R; Hossain, Khwaja; Kalavacharla, Venu

    2016-01-01

    Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar ("Sierra") using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation.

  5. Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing

    PubMed Central

    Crampton, Mollee; Sripathi, Venkateswara R.; Hossain, Khwaja; Kalavacharla, Venu

    2016-01-01

    Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar (“Sierra”) using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation. PMID:27199997

  6. High glucose recovery from direct enzymatic hydrolysis of bisulfite-pretreatment on non-detoxified furfural residues.

    PubMed

    Xing, Yang; Bu, Lingxi; Sun, Dafeng; Liu, Zhiping; Liu, Shijie; Jiang, Jianxin

    2015-10-01

    This study reports four schemes to pretreat wet furfural residues (FRs) with sodium bisulfite for production of fermentable sugar. The results showed that non-detoxified FRs (pH 2-3) had great potential to lower the cost of bioconversion. The optimal process was that unwashed FRs were first pretreated with bisulfite, and the whole slurry was then directly used for enzymatic hydrolysis. A maximum glucose yield of 99.4% was achieved from substrates pretreated with 0.1 g NaHSO3/g dry substrate (DS), at a relatively low temperature of 100 °C for 3 h. Compared with raw material, enzymatic hydrolysis at a high-solid of 16.5% (w/w) specifically showed more excellent performance with bisulfite treated FRs. Direct bisulfite pretreatment improved the accessibility of substrates and the total glucose recovery. Lignosulfonate in the non-detoxified slurry decreased the non-productive adsorption of cellulase on the substrate, thus improving enzymatic hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Validation of a stability-indicating hydrophilic interaction liquid chromatographic method for the quantitative determination of vitamin k3 (menadione sodium bisulfite) in injectable solution formulation.

    PubMed

    Ghanem, Mashhour M; Abu-Lafi, Saleh A; Hallak, Hussein O

    2013-01-01

    A simple, specific, accurate, and stability-indicating method was developed and validated for the quantitative determination of menadione sodium bisulfite in the injectable solution formulation. The method is based on zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) coupled with a photodiode array detector. The desired separation was achieved on the ZIC-HILIC column (250 mm × 4.6 mm, 5 μm) at 25°C temperature. The optimized mobile phase consisted of an isocratic solvent mixture of 200mM ammonium acetate (NH4AC) solution and acetonitrile (ACN) (20:80; v/v) pH-adjusted to 5.7 by glacial acetic acid. The mobile phase was fixed at 0.5 ml/min and the analytes were monitored at 261 nm using a photodiode array detector. The effects of the chromatographic conditions on the peak retention, peak USP tailing factor, and column efficiency were systematically optimized. Forced degradation experiments were carried out by exposing menadione sodium bisulfite standard and the injectable solution formulation to thermal, photolytic, oxidative, and acid-base hydrolytic stress conditions. The degradation products were well-resolved from the main peak and the excipients, thus proving that the method is a reliable, stability-indicating tool. The method was validated as per ICH and USP guidelines (USP34/NF29) and found to be adequate for the routine quantitative estimation of menadione sodium bisulfite in commercially available menadione sodium bisulfite injectable solution dosage forms.

  8. Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

    PubMed Central

    Ojha, Sunil; Watson, Douglas S.; Bomar, Martha G.; Galande, Amit K.; Shearer, Alexander G.

    2013-01-01

    The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation. PMID:24386392

  9. Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

    PubMed

    Ramkissoon, Kevin R; Miller, Jennifer K; Ojha, Sunil; Watson, Douglas S; Bomar, Martha G; Galande, Amit K; Shearer, Alexander G

    2013-01-01

    The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

  10. Phylogenetic and environmental diversity of DsrAB-type dissimilatory (bi)sulfite reductases

    PubMed Central

    Müller, Albert Leopold; Kjeldsen, Kasper Urup; Rattei, Thomas; Pester, Michael; Loy, Alexander

    2015-01-01

    The energy metabolism of essential microbial guilds in the biogeochemical sulfur cycle is based on a DsrAB-type dissimilatory (bi)sulfite reductase that either catalyzes the reduction of sulfite to sulfide during anaerobic respiration of sulfate, sulfite and organosulfonates, or acts in reverse during sulfur oxidation. Common use of dsrAB as a functional marker showed that dsrAB richness in many environments is dominated by novel sequence variants and collectively represents an extensive, largely uncharted sequence assemblage. Here, we established a comprehensive, manually curated dsrAB/DsrAB database and used it to categorize the known dsrAB diversity, reanalyze the evolutionary history of dsrAB and evaluate the coverage of published dsrAB-targeted primers. Based on a DsrAB consensus phylogeny, we introduce an operational classification system for environmental dsrAB sequences that integrates established taxonomic groups with operational taxonomic units (OTUs) at multiple phylogenetic levels, ranging from DsrAB enzyme families that reflect reductive or oxidative DsrAB types of bacterial or archaeal origin, superclusters, uncultured family-level lineages to species-level OTUs. Environmental dsrAB sequences constituted at least 13 stable family-level lineages without any cultivated representatives, suggesting that major taxa of sulfite/sulfate-reducing microorganisms have not yet been identified. Three of these uncultured lineages occur mainly in marine environments, while specific habitat preferences are not evident for members of the other 10 uncultured lineages. In summary, our publically available dsrAB/DsrAB database, the phylogenetic framework, the multilevel classification system and a set of recommended primers provide a necessary foundation for large-scale dsrAB ecology studies with next-generation sequencing methods. PMID:25343514

  11. Influence of 2,3-diphosphoglycerate metabolism on sodium-potassium permeability in human red blood cells: studies with bisulfite and other redox agents

    PubMed Central

    Parker, John C.

    1969-01-01

    It is known that bisulfite ions can selectively deplete red blood cells of 2,3-diphosphoglycerate (2,3-DPG). Studies of the effects of bisulfite on sodium-potassium permeability and metabolism were undertaken to clarify the physiologic role of the abundant quantities of 2,3-DPG in human erythrocytes. Treatment of cells with bisulfite results in a reversible increase in the passive permeability to Na and K ions. Metabolism of glucose to lactate is increased, with a rise in the intracellular ratio of fructose diphosphate to hexose monophosphate. Cell 2,3-DPG is quantitatively converted to pyruvate and inorganic phosphate. The permeability effects of bisulfite are countered by ethacrynic acid and by such oxidizing agents as pyruvate and methylene blue. Taken together, the results suggest that the effects on Na-K flux of bisulfite are related more to the reducing potential of this anion than to its capacity to deplete cells of 2,3-DPG. PMID:5765015

  12. SCPRED: accurate prediction of protein structural class for sequences of twilight-zone similarity with predicting sequences.

    PubMed

    Kurgan, Lukasz; Cios, Krzysztof; Chen, Ke

    2008-05-01

    Protein structure prediction methods provide accurate results when a homologous protein is predicted, while poorer predictions are obtained in the absence of homologous templates. However, some protein chains that share twilight-zone pairwise identity can form similar folds and thus determining structural similarity without the sequence similarity would be desirable for the structure prediction. The folding type of a protein or its domain is defined as the structural class. Current structural class prediction methods that predict the four structural classes defined in SCOP provide up to 63% accuracy for the datasets in which sequence identity of any pair of sequences belongs to the twilight-zone. We propose SCPRED method that improves prediction accuracy for sequences that share twilight-zone pairwise similarity with sequences used for the prediction. SCPRED uses a support vector machine classifier that takes several custom-designed features as its input to predict the structural classes. Based on extensive design that considers over 2300 index-, composition- and physicochemical properties-based features along with features based on the predicted secondary structure and content, the classifier's input includes 8 features based on information extracted from the secondary structure predicted with PSI-PRED and one feature computed from the sequence. Tests performed with datasets of 1673 protein chains, in which any pair of sequences shares twilight-zone similarity, show that SCPRED obtains 80.3% accuracy when predicting the four SCOP-defined structural classes, which is superior when compared with over a dozen recent competing methods that are based on support vector machine, logistic regression, and ensemble of classifiers predictors. The SCPRED can accurately find similar structures for sequences that share low identity with sequence used for the prediction. The high predictive accuracy achieved by SCPRED is attributed to the design of the features, which are

  13. SCPRED: Accurate prediction of protein structural class for sequences of twilight-zone similarity with predicting sequences

    PubMed Central

    Kurgan, Lukasz; Cios, Krzysztof; Chen, Ke

    2008-01-01

    Background Protein structure prediction methods provide accurate results when a homologous protein is predicted, while poorer predictions are obtained in the absence of homologous templates. However, some protein chains that share twilight-zone pairwise identity can form similar folds and thus determining structural similarity without the sequence similarity would be desirable for the structure prediction. The folding type of a protein or its domain is defined as the structural class. Current structural class prediction methods that predict the four structural classes defined in SCOP provide up to 63% accuracy for the datasets in which sequence identity of any pair of sequences belongs to the twilight-zone. We propose SCPRED method that improves prediction accuracy for sequences that share twilight-zone pairwise similarity with sequences used for the prediction. Results SCPRED uses a support vector machine classifier that takes several custom-designed features as its input to predict the structural classes. Based on extensive design that considers over 2300 index-, composition- and physicochemical properties-based features along with features based on the predicted secondary structure and content, the classifier's input includes 8 features based on information extracted from the secondary structure predicted with PSI-PRED and one feature computed from the sequence. Tests performed with datasets of 1673 protein chains, in which any pair of sequences shares twilight-zone similarity, show that SCPRED obtains 80.3% accuracy when predicting the four SCOP-defined structural classes, which is superior when compared with over a dozen recent competing methods that are based on support vector machine, logistic regression, and ensemble of classifiers predictors. Conclusion The SCPRED can accurately find similar structures for sequences that share low identity with sequence used for the prediction. The high predictive accuracy achieved by SCPRED is attributed to the design of

  14. Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

    PubMed

    Reid-Bayliss, Kate S; Loeb, Lawrence A

    2017-08-29

    Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

  15. Attachment of reporter groups to specific, selected cytidine residues in RNA using a bisulfite-catalyzed transamination reaction.

    PubMed Central

    Draper, D E

    1984-01-01

    Bisulfite catalyzes transamination of cytidine at the N4 position; the suitability of this reaction for attaching reporter groups to selected cytidine residues in RNA molecules has been investigated. Poly(C) is nearly quantitatively converted to the poly (N4 aminoethyl-C) derivative after 3 hrs at 42 degrees C with ethylene diamine (pK1 = 7.6) and bisulfite. This derivative reacts quantitatively with N-hydroxysuccinimide esters; the linkage of a fluorescent dye, nitrobenzofurazan, to cytidine by this reaction is demonstrated. To direct the bisulfite reaction to selected cytidines within a large RNA molecule, the RNA is hybridized to complementary DNA containing a deletion. Only the cytidines in the single strand RNA loop (corresponding to the DNA deletion) are reactive. Two cytidines in the middle of a 340 base RNA fragment from 16S ribosomal RNA have been modified by this technique. Images PMID:6198634

  16. Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver

    PubMed Central

    Blanquart, François; Golubchik, Tanya; Gall, Astrid; Bakker, Margreet; Bezemer, Daniela; Croucher, Nicholas J; Hall, Matthew; Hillebregt, Mariska; Ratmann, Oliver; Albert, Jan; Bannert, Norbert; Fellay, Jacques; Fransen, Katrien; Gourlay, Annabelle; Grabowski, M Kate; Gunsenheimer-Bartmeyer, Barbara; Günthard, Huldrych F; Kivelä, Pia; Kouyos, Roger; Laeyendecker, Oliver; Liitsola, Kirsi; Meyer, Laurence; Porter, Kholoud; Ristola, Matti; van Sighem, Ard; Cornelissen, Marion; Kellam, Paul; Reiss, Peter

    2018-01-01

    Abstract Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user’s choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver’s constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also

  17. Easy and accurate reconstruction of whole HIV genomes from short-read sequence data with shiver.

    PubMed

    Wymant, Chris; Blanquart, François; Golubchik, Tanya; Gall, Astrid; Bakker, Margreet; Bezemer, Daniela; Croucher, Nicholas J; Hall, Matthew; Hillebregt, Mariska; Ong, Swee Hoe; Ratmann, Oliver; Albert, Jan; Bannert, Norbert; Fellay, Jacques; Fransen, Katrien; Gourlay, Annabelle; Grabowski, M Kate; Gunsenheimer-Bartmeyer, Barbara; Günthard, Huldrych F; Kivelä, Pia; Kouyos, Roger; Laeyendecker, Oliver; Liitsola, Kirsi; Meyer, Laurence; Porter, Kholoud; Ristola, Matti; van Sighem, Ard; Berkhout, Ben; Cornelissen, Marion; Kellam, Paul; Reiss, Peter; Fraser, Christophe

    2018-01-01

    Studying the evolution of viruses and their molecular epidemiology relies on accurate viral sequence data, so that small differences between similar viruses can be meaningfully interpreted. Despite its higher throughput and more detailed minority variant data, next-generation sequencing has yet to be widely adopted for HIV. The difficulty of accurately reconstructing the consensus sequence of a quasispecies from reads (short fragments of DNA) in the presence of large between- and within-host diversity, including frequent indels, may have presented a barrier. In particular, mapping (aligning) reads to a reference sequence leads to biased loss of information; this bias can distort epidemiological and evolutionary conclusions. De novo assembly avoids this bias by aligning the reads to themselves, producing a set of sequences called contigs. However contigs provide only a partial summary of the reads, misassembly may result in their having an incorrect structure, and no information is available at parts of the genome where contigs could not be assembled. To address these problems we developed the tool shiver to pre-process reads for quality and contamination, then map them to a reference tailored to the sample using corrected contigs supplemented with the user's choice of existing reference sequences. Run with two commands per sample, it can easily be used for large heterogeneous data sets. We used shiver to reconstruct the consensus sequence and minority variant information from paired-end short-read whole-genome data produced with the Illumina platform, for sixty-five existing publicly available samples and fifty new samples. We show the systematic superiority of mapping to shiver's constructed reference compared with mapping the same reads to the closest of 3,249 real references: median values of 13 bases called differently and more accurately, 0 bases called differently and less accurately, and 205 bases of missing sequence recovered. We also successfully applied

  18. SATe-II: very fast and accurate simultaneous estimation of multiple sequence alignments and phylogenetic trees.

    PubMed

    Liu, Kevin; Warnow, Tandy J; Holder, Mark T; Nelesen, Serita M; Yu, Jiaye; Stamatakis, Alexandros P; Linder, C Randal

    2012-01-01

    Highly accurate estimation of phylogenetic trees for large data sets is difficult, in part because multiple sequence alignments must be accurate for phylogeny estimation methods to be accurate. Coestimation of alignments and trees has been attempted but currently only SATé estimates reasonably accurate trees and alignments for large data sets in practical time frames (Liu K., Raghavan S., Nelesen S., Linder C.R., Warnow T. 2009b. Rapid and accurate large-scale coestimation of sequence alignments and phylogenetic trees. Science. 324:1561-1564). Here, we present a modification to the original SATé algorithm that improves upon SATé (which we now call SATé-I) in terms of speed and of phylogenetic and alignment accuracy. SATé-II uses a different divide-and-conquer strategy than SATé-I and so produces smaller more closely related subsets than SATé-I; as a result, SATé-II produces more accurate alignments and trees, can analyze larger data sets, and runs more efficiently than SATé-I. Generally, SATé is a metamethod that takes an existing multiple sequence alignment method as an input parameter and boosts the quality of that alignment method. SATé-II-boosted alignment methods are significantly more accurate than their unboosted versions, and trees based upon these improved alignments are more accurate than trees based upon the original alignments. Because SATé-I used maximum likelihood (ML) methods that treat gaps as missing data to estimate trees and because we found a correlation between the quality of tree/alignment pairs and ML scores, we explored the degree to which SATé's performance depends on using ML with gaps treated as missing data to determine the best tree/alignment pair. We present two lines of evidence that using ML with gaps treated as missing data to optimize the alignment and tree produces very poor results. First, we show that the optimization problem where a set of unaligned DNA sequences is given and the output is the tree and alignment of

  19. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.

  20. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed Central

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-01-01

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  1. Experimental estimation of the bisulfite isomer quotient as a function of temperature: Implications for sulfur isotope fractionations in aqueous sulfite solutions

    NASA Astrophysics Data System (ADS)

    Eldridge, Daniel L.; Mysen, Bjorn O.; Cody, George D.

    2018-01-01

    Bisulfite (HSO3-) and sulfite (SO32-) compounds play key roles in numerous geochemical and biochemical processes extending from the atmosphere to the subseafloor biosphere. Despite decades of spectroscopic investigations, the molecular composition of HSO3- in solution remains uncertain and, thus, the role of bisulfite in (bio)chemical and isotope fractionation processes is unclear. We report new experimental estimates for the bisulfite isomer quotient (Qi = [(HO)SO2-]/[(HS)O3-]; [] = concentration) as a function of temperature from the interpretation of Raman spectra collected from aqueous NaHSO3 solutions contained in fused silica capsules. In pure NaHSO3 solutions (1Na+:1HSO3-, stoichiometric) over [NaHSO3] = 0.2-0.4 m (moles/kg H2O), the following relationship is obtained:

  2. A new fluorescent enhanced probe based on (E)-9-(2-nitrovinyl)-anthracene for the detection of bisulfite anions and its practical application

    NASA Astrophysics Data System (ADS)

    Chao, Jianbin; Liu, Yuhong; Zhang, Yan; Zhang, Yongbin; Huo, Fangjun; Yin, Caixia; Wang, Yu; Qin, Liping

    2015-07-01

    A new fluorescent enhanced probe based on (E)-9-(2-nitrovinyl)-anthracene is developed, which shows high selectivity and sensitivity for the detection of bisulfite anions at Na2HPO4 citric acid buffer solutions (pH 5.0). When addition of HSO3-, the fluorescence intensity is significantly enhanced and the probe displays apparent fluorescence color changes from non-fluorescence to blue under a UV lamp illumination, the solution color also changes from yellow to colorless. The detection limit is determined to be as low as 6.30 μM. This offers another specific colorimetric and fluorescent probe for bisulfite anions detection, furthermore it is applied in detecting the level of bisulfite in sugar samples.

  3. Flexible, fast and accurate sequence alignment profiling on GPGPU with PaSWAS.

    PubMed

    Warris, Sven; Yalcin, Feyruz; Jackson, Katherine J L; Nap, Jan Peter

    2015-01-01

    To obtain large-scale sequence alignments in a fast and flexible way is an important step in the analyses of next generation sequencing data. Applications based on the Smith-Waterman (SW) algorithm are often either not fast enough, limited to dedicated tasks or not sufficiently accurate due to statistical issues. Current SW implementations that run on graphics hardware do not report the alignment details necessary for further analysis. With the Parallel SW Alignment Software (PaSWAS) it is possible (a) to have easy access to the computational power of NVIDIA-based general purpose graphics processing units (GPGPUs) to perform high-speed sequence alignments, and (b) retrieve relevant information such as score, number of gaps and mismatches. The software reports multiple hits per alignment. The added value of the new SW implementation is demonstrated with two test cases: (1) tag recovery in next generation sequence data and (2) isotype assignment within an immunoglobulin 454 sequence data set. Both cases show the usability and versatility of the new parallel Smith-Waterman implementation.

  4. Fast, accurate and easy-to-pipeline methods for amplicon sequence processing

    NASA Astrophysics Data System (ADS)

    Antonielli, Livio; Sessitsch, Angela

    2016-04-01

    Next generation sequencing (NGS) technologies established since years as an essential resource in microbiology. While on the one hand metagenomic studies can benefit from the continuously increasing throughput of the Illumina (Solexa) technology, on the other hand the spreading of third generation sequencing technologies (PacBio, Oxford Nanopore) are getting whole genome sequencing beyond the assembly of fragmented draft genomes, making it now possible to finish bacterial genomes even without short read correction. Besides (meta)genomic analysis next-gen amplicon sequencing is still fundamental for microbial studies. Amplicon sequencing of the 16S rRNA gene and ITS (Internal Transcribed Spacer) remains a well-established widespread method for a multitude of different purposes concerning the identification and comparison of archaeal/bacterial (16S rRNA gene) and fungal (ITS) communities occurring in diverse environments. Numerous different pipelines have been developed in order to process NGS-derived amplicon sequences, among which Mothur, QIIME and USEARCH are the most well-known and cited ones. The entire process from initial raw sequence data through read error correction, paired-end read assembly, primer stripping, quality filtering, clustering, OTU taxonomic classification and BIOM table rarefaction as well as alternative "normalization" methods will be addressed. An effective and accurate strategy will be presented using the state-of-the-art bioinformatic tools and the example of a straightforward one-script pipeline for 16S rRNA gene or ITS MiSeq amplicon sequencing will be provided. Finally, instructions on how to automatically retrieve nucleotide sequences from NCBI and therefore apply the pipeline to targets other than 16S rRNA gene (Greengenes, SILVA) and ITS (UNITE) will be discussed.

  5. Metagenomic Analysis Indicates Epsilonproteobacteria as a Potential Cause of Microbial Corrosion in Pipelines Injected with Bisulfite

    PubMed Central

    An, Dongshan; Dong, Xiaoli; An, Annie; Park, Hyung S.; Strous, Marc; Voordouw, Gerrit

    2016-01-01

    Sodium bisulfite (SBS) is used as an oxygen scavenger to decrease corrosion in pipelines transporting brackish subsurface water used in the production of bitumen by steam-assisted gravity drainage. Sequencing 16S rRNA gene amplicons has indicated that SBS addition increased the fraction of the sulfate-reducing bacteria (SRB) Desulfomicrobium, as well as of Desulfocapsa, which can also grow by disproportionating sulfite into sulfide, sulfur, and sulfate. SRB use cathodic H2, formed by reduction of aqueous protons at the iron surface, or use low potential electrons from iron and aqueous protons directly for sulfate reduction. In order to reveal the effects of SBS treatment in more detail, metagenomic analysis was performed with pipe-associated solids (PAS) scraped from a pipe section upstream (PAS-616P) and downstream (PAS-821TP) of the SBS injection point. A major SBS-induced change in microbial community composition and in affiliated hynL genes for the large subunit of [NiFe] hydrogenase was the appearance of sulfur-metabolizing Epsilonproteobacteria of the genera Sulfuricurvum and Sulfurovum. These are chemolithotrophs, which oxidize sulfide or sulfur with O2 or reduce sulfur with H2. Because O2 was absent, this class likely catalyzed reduction of sulfur (S0) originating from the metabolism of bisulfite with cathodic H2 (or low potential electrons and aqueous protons) originating from the corrosion of steel (Fe0). Overall this accelerates reaction of of S0 and Fe0 to form FeS, making this class a potentially powerful contributor to microbial corrosion. The PAS-821TP metagenome also had increased fractions of Deltaproteobacteria including the SRB Desulfomicrobium and Desulfocapsa. Altogether, SBS increased the fraction of hydrogen-utilizing Delta- and Epsilonproteobacteria in brackish-water-transporting pipelines, potentially stimulating anaerobic pipeline corrosion if dosed in excess of the intended oxygen scavenger function. PMID:26858705

  6. A Novel Method for Accurate Operon Predictions in All SequencedProkaryotes

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

    Price, Morgan N.; Huang, Katherine H.; Alm, Eric J.

    2004-12-01

    We combine comparative genomic measures and the distance separating adjacent genes to predict operons in 124 completely sequenced prokaryotic genomes. Our method automatically tailors itself to each genome using sequence information alone, and thus can be applied to any prokaryote. For Escherichia coli K12 and Bacillus subtilis, our method is 85 and 83% accurate, respectively, which is similar to the accuracy of methods that use the same features but are trained on experimentally characterized transcripts. In Halobacterium NRC-1 and in Helicobacterpylori, our method correctly infers that genes in operons are separated by shorter distances than they are in E.coli, andmore » its predictions using distance alone are more accurate than distance-only predictions trained on a database of E.coli transcripts. We use microarray data from sixphylogenetically diverse prokaryotes to show that combining intergenic distance with comparative genomic measures further improves accuracy and that our method is broadly effective. Finally, we survey operon structure across 124 genomes, and find several surprises: H.pylori has many operons, contrary to previous reports; Bacillus anthracis has an unusual number of pseudogenes within conserved operons; and Synechocystis PCC6803 has many operons even though it has unusually wide spacings between conserved adjacent genes.« less

  7. MOSAIK: a hash-based algorithm for accurate next-generation sequencing short-read mapping.

    PubMed

    Lee, Wan-Ping; Stromberg, Michael P; Ward, Alistair; Stewart, Chip; Garrison, Erik P; Marth, Gabor T

    2014-01-01

    MOSAIK is a stable, sensitive and open-source program for mapping second and third-generation sequencing reads to a reference genome. Uniquely among current mapping tools, MOSAIK can align reads generated by all the major sequencing technologies, including Illumina, Applied Biosystems SOLiD, Roche 454, Ion Torrent and Pacific BioSciences SMRT. Indeed, MOSAIK was the only aligner to provide consistent mappings for all the generated data (sequencing technologies, low-coverage and exome) in the 1000 Genomes Project. To provide highly accurate alignments, MOSAIK employs a hash clustering strategy coupled with the Smith-Waterman algorithm. This method is well-suited to capture mismatches as well as short insertions and deletions. To support the growing interest in larger structural variant (SV) discovery, MOSAIK provides explicit support for handling known-sequence SVs, e.g. mobile element insertions (MEIs) as well as generating outputs tailored to aid in SV discovery. All variant discovery benefits from an accurate description of the read placement confidence. To this end, MOSAIK uses a neural-network based training scheme to provide well-calibrated mapping quality scores, demonstrated by a correlation coefficient between MOSAIK assigned and actual mapping qualities greater than 0.98. In order to ensure that studies of any genome are supported, a training pipeline is provided to ensure optimal mapping quality scores for the genome under investigation. MOSAIK is multi-threaded, open source, and incorporated into our command and pipeline launcher system GKNO (http://gkno.me).

  8. MOSAIK: A Hash-Based Algorithm for Accurate Next-Generation Sequencing Short-Read Mapping

    PubMed Central

    Lee, Wan-Ping; Stromberg, Michael P.; Ward, Alistair; Stewart, Chip; Garrison, Erik P.; Marth, Gabor T.

    2014-01-01

    MOSAIK is a stable, sensitive and open-source program for mapping second and third-generation sequencing reads to a reference genome. Uniquely among current mapping tools, MOSAIK can align reads generated by all the major sequencing technologies, including Illumina, Applied Biosystems SOLiD, Roche 454, Ion Torrent and Pacific BioSciences SMRT. Indeed, MOSAIK was the only aligner to provide consistent mappings for all the generated data (sequencing technologies, low-coverage and exome) in the 1000 Genomes Project. To provide highly accurate alignments, MOSAIK employs a hash clustering strategy coupled with the Smith-Waterman algorithm. This method is well-suited to capture mismatches as well as short insertions and deletions. To support the growing interest in larger structural variant (SV) discovery, MOSAIK provides explicit support for handling known-sequence SVs, e.g. mobile element insertions (MEIs) as well as generating outputs tailored to aid in SV discovery. All variant discovery benefits from an accurate description of the read placement confidence. To this end, MOSAIK uses a neural-network based training scheme to provide well-calibrated mapping quality scores, demonstrated by a correlation coefficient between MOSAIK assigned and actual mapping qualities greater than 0.98. In order to ensure that studies of any genome are supported, a training pipeline is provided to ensure optimal mapping quality scores for the genome under investigation. MOSAIK is multi-threaded, open source, and incorporated into our command and pipeline launcher system GKNO (http://gkno.me). PMID:24599324

  9. Accurate prediction of protein–protein interactions from sequence alignments using a Bayesian method

    PubMed Central

    Burger, Lukas; van Nimwegen, Erik

    2008-01-01

    Accurate and large-scale prediction of protein–protein interactions directly from amino-acid sequences is one of the great challenges in computational biology. Here we present a new Bayesian network method that predicts interaction partners using only multiple alignments of amino-acid sequences of interacting protein domains, without tunable parameters, and without the need for any training examples. We first apply the method to bacterial two-component systems and comprehensively reconstruct two-component signaling networks across all sequenced bacteria. Comparisons of our predictions with known interactions show that our method infers interaction partners genome-wide with high accuracy. To demonstrate the general applicability of our method we show that it also accurately predicts interaction partners in a recent dataset of polyketide synthases. Analysis of the predicted genome-wide two-component signaling networks shows that cognates (interacting kinase/regulator pairs, which lie adjacent on the genome) and orphans (which lie isolated) form two relatively independent components of the signaling network in each genome. In addition, while most genes are predicted to have only a small number of interaction partners, we find that 10% of orphans form a separate class of ‘hub' nodes that distribute and integrate signals to and from up to tens of different interaction partners. PMID:18277381

  10. A Bayesian hierarchical model to detect differentially methylated loci from single nucleotide resolution sequencing data

    PubMed Central

    Feng, Hao; Conneely, Karen N.; Wu, Hao

    2014-01-01

    DNA methylation is an important epigenetic modification that has essential roles in cellular processes including gene regulation, development and disease and is widely dysregulated in most types of cancer. Recent advances in sequencing technology have enabled the measurement of DNA methylation at single nucleotide resolution through methods such as whole-genome bisulfite sequencing and reduced representation bisulfite sequencing. In DNA methylation studies, a key task is to identify differences under distinct biological contexts, for example, between tumor and normal tissue. A challenge in sequencing studies is that the number of biological replicates is often limited by the costs of sequencing. The small number of replicates leads to unstable variance estimation, which can reduce accuracy to detect differentially methylated loci (DML). Here we propose a novel statistical method to detect DML when comparing two treatment groups. The sequencing counts are described by a lognormal-beta-binomial hierarchical model, which provides a basis for information sharing across different CpG sites. A Wald test is developed for hypothesis testing at each CpG site. Simulation results show that the proposed method yields improved DML detection compared to existing methods, particularly when the number of replicates is low. The proposed method is implemented in the Bioconductor package DSS. PMID:24561809

  11. Enhancement of high-solids enzymatic hydrolysis of corncob residues by bisulfite pretreatment for biorefinery.

    PubMed

    Xing, Yang; Bu, Lingxi; Zheng, Tianran; Liu, Shijie; Jiang, Jianxin

    2016-12-01

    Co-production of glucose, furfural and other green materials based on a lignocellulosic biorefinery is a promising way to realize the commercial application of corncob residues. An effective process was developed for glucose production using low temperature bisulfite pretreatment and high-solids enzymatic hydrolysis. Corncob residues from furfural production (FRs) were pretreated with 0.1g NaHSO 3 /g dry substrate at 100°C for 3h. Lignin was sulfonated and sulfonic groups were produced during pretreatment, which resulted in decreasing the zeta potential of the samples. Compared with raw material, bisulfite pretreatment of FRs increased the glucose yield from 18.6 to 99.45% after 72h hydrolysis at a solids loading of 12.5%. The hydrolysis residues showed a relatively high thermal stability and concentrated high derivatives. Direct pretreatment followed by enzymatic hydrolysis is an environmentally-friendly and economically-feasible method for the production of glucose and high-purity lignin, which could be further converted into high-value products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Accurate Sample Assignment in a Multiplexed, Ultrasensitive, High-Throughput Sequencing Assay for Minimal Residual Disease.

    PubMed

    Bartram, Jack; Mountjoy, Edward; Brooks, Tony; Hancock, Jeremy; Williamson, Helen; Wright, Gary; Moppett, John; Goulden, Nick; Hubank, Mike

    2016-07-01

    High-throughput sequencing (HTS) (next-generation sequencing) of the rearranged Ig and T-cell receptor genes promises to be less expensive and more sensitive than current methods of monitoring minimal residual disease (MRD) in patients with acute lymphoblastic leukemia. However, the adoption of new approaches by clinical laboratories requires careful evaluation of all potential sources of error and the development of strategies to ensure the highest accuracy. Timely and efficient clinical use of HTS platforms will depend on combining multiple samples (multiplexing) in each sequencing run. Here we examine the Ig heavy-chain gene HTS on the Illumina MiSeq platform for MRD. We identify errors associated with multiplexing that could potentially impact the accuracy of MRD analysis. We optimize a strategy that combines high-purity, sequence-optimized oligonucleotides, dual indexing, and an error-aware demultiplexing approach to minimize errors and maximize sensitivity. We present a probability-based, demultiplexing pipeline Error-Aware Demultiplexer that is suitable for all MiSeq strategies and accurately assigns samples to the correct identifier without excessive loss of data. Finally, using controls quantified by digital PCR, we show that HTS-MRD can accurately detect as few as 1 in 10(6) copies of specific leukemic MRD. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  13. Statistical method evaluation for differentially methylated CpGs in base resolution next-generation DNA sequencing data.

    PubMed

    Zhang, Yun; Baheti, Saurabh; Sun, Zhifu

    2018-05-01

    High-throughput bisulfite methylation sequencing such as reduced representation bisulfite sequencing (RRBS), Agilent SureSelect Human Methyl-Seq (Methyl-seq) or whole-genome bisulfite sequencing is commonly used for base resolution methylome research. These data are represented either by the ratio of methylated cytosine versus total coverage at a CpG site or numbers of methylated and unmethylated cytosines. Multiple statistical methods can be used to detect differentially methylated CpGs (DMCs) between conditions, and these methods are often the base for the next step of differentially methylated region identification. The ratio data have a flexibility of fitting to many linear models, but the raw count data take consideration of coverage information. There is an array of options in each datatype for DMC detection; however, it is not clear which is an optimal statistical method. In this study, we systematically evaluated four statistic methods on methylation ratio data and four methods on count-based data and compared their performances with regard to type I error control, sensitivity and specificity of DMC detection and computational resource demands using real RRBS data along with simulation. Our results show that the ratio-based tests are generally more conservative (less sensitive) than the count-based tests. However, some count-based methods have high false-positive rates and should be avoided. The beta-binomial model gives a good balance between sensitivity and specificity and is preferred method. Selection of methods in different settings, signal versus noise and sample size estimation are also discussed.

  14. Resin in bisulfite pulp from Pinus radiata wood and its relationship to pitch troubles

    Treesearch

    P.J. Nelson; Richard W. Hemingway

    1971-01-01

    The resin content of bisulfite pulp from Pinus radiata D. Don was determined at various stages in its manufacture and the changes in the composition of the resin studied. About 50% of the resin present in the wood was removed during cooking, an additional 11% by blowpit washing, and a further 8% by screening. Resin acids and fatty acids were...

  15. The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis.

    PubMed

    Tuorto, Francesca; Herbst, Friederike; Alerasool, Nader; Bender, Sebastian; Popp, Oliver; Federico, Giuseppina; Reitter, Sonja; Liebers, Reinhard; Stoecklin, Georg; Gröne, Hermann-Josef; Dittmar, Gunnar; Glimm, Hanno; Lyko, Frank

    2015-09-14

    The Dnmt2 enzyme utilizes the catalytic mechanism of eukaryotic DNA methyltransferases to methylate several tRNAs at cytosine 38. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile, and the biological function of Dnmt2 has remained elusive. Here, we show that endochondral ossification is delayed in newborn Dnmt2-deficient mice, which is accompanied by a reduction of the haematopoietic stem and progenitor cell population and a cell-autonomous defect in their differentiation. RNA bisulfite sequencing revealed that Dnmt2 methylates C38 of tRNA Asp(GTC), Gly(GCC), and Val(AAC), thus preventing tRNA fragmentation. Proteomic analyses from primary bone marrow cells uncovered systematic differences in protein expression that are due to specific codon mistranslation by tRNAs lacking Dnmt2-dependent methylation. Our observations demonstrate that Dnmt2 plays an important role in haematopoiesis and define a novel function of C38 tRNA methylation in the discrimination of near-cognate codons, thereby ensuring accurate polypeptide synthesis. © 2015 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  16. Effect of Sodium Bisulfite Injection on the Microbial Community Composition in a Brackish-Water-Transporting Pipeline▿†

    PubMed Central

    Park, Hyung Soo; Chatterjee, Indranil; Dong, Xiaoli; Wang, Sheng-Hung; Sensen, Christoph W.; Caffrey, Sean M.; Jack, Thomas R.; Boivin, Joe; Voordouw, Gerrit

    2011-01-01

    Pipelines transporting brackish subsurface water, used in the production of bitumen by steam-assisted gravity drainage, are subject to frequent corrosion failures despite the addition of the oxygen scavenger sodium bisulfite (SBS). Pyrosequencing of 16S rRNA genes was used to determine the microbial community composition for planktonic samples of transported water and for sessile samples of pipe-associated solids (PAS) scraped from pipeline cutouts representing corrosion failures. These were obtained from upstream (PAS-616P) and downstream (PAS-821TP and PAS-821LP, collected under rapid-flow and stagnant conditions, respectively) of the SBS injection point. Most transported water samples had a large fraction (1.8% to 97% of pyrosequencing reads) of Pseudomonas not found in sessile pipe samples. The sessile population of PAS-616P had methanogens (Methanobacteriaceae) as the main (56%) community component, whereas Deltaproteobacteria of the genera Desulfomicrobium and Desulfocapsa were not detected. In contrast, PAS-821TP and PAS-821LP had lower fractions (41% and 0.6%) of Methanobacteriaceae archaea but increased fractions of sulfate-reducing Desulfomicrobium (18% and 48%) and of bisulfite-disproportionating Desulfocapsa (35% and 22%) bacteria. Hence, SBS injection strongly changed the sessile microbial community populations. X-ray diffraction analysis of pipeline scale indicated that iron carbonate was present both upstream and downstream, whereas iron sulfide and sulfur were found only downstream of the SBS injection point, suggesting a contribution of the bisulfite-disproportionating and sulfate-reducing bacteria in the scale to iron corrosion. Incubation of iron coupons with pipeline waters indicated iron corrosion coupled to the formation of methane. Hence, both methanogenic and sulfidogenic microbial communities contributed to corrosion of pipelines transporting these brackish waters. PMID:21856836

  17. Accurate multiple sequence-structure alignment of RNA sequences using combinatorial optimization.

    PubMed

    Bauer, Markus; Klau, Gunnar W; Reinert, Knut

    2007-07-27

    The discovery of functional non-coding RNA sequences has led to an increasing interest in algorithms related to RNA analysis. Traditional sequence alignment algorithms, however, fail at computing reliable alignments of low-homology RNA sequences. The spatial conformation of RNA sequences largely determines their function, and therefore RNA alignment algorithms have to take structural information into account. We present a graph-based representation for sequence-structure alignments, which we model as an integer linear program (ILP). We sketch how we compute an optimal or near-optimal solution to the ILP using methods from combinatorial optimization, and present results on a recently published benchmark set for RNA alignments. The implementation of our algorithm yields better alignments in terms of two published scores than the other programs that we tested: This is especially the case with an increasing number of input sequences. Our program LARA is freely available for academic purposes from http://www.planet-lisa.net.

  18. Use of Whole-Genus Genome Sequence Data To Develop a Multilocus Sequence Typing Tool That Accurately Identifies Yersinia Isolates to the Species and Subspecies Levels

    PubMed Central

    Hall, Miquette; Chattaway, Marie A.; Reuter, Sandra; Savin, Cyril; Strauch, Eckhard; Carniel, Elisabeth; Connor, Thomas; Van Damme, Inge; Rajakaruna, Lakshani; Rajendram, Dunstan; Jenkins, Claire; Thomson, Nicholas R.

    2014-01-01

    The genus Yersinia is a large and diverse bacterial genus consisting of human-pathogenic species, a fish-pathogenic species, and a large number of environmental species. Recently, the phylogenetic and population structure of the entire genus was elucidated through the genome sequence data of 241 strains encompassing every known species in the genus. Here we report the mining of this enormous data set to create a multilocus sequence typing-based scheme that can identify Yersinia strains to the species level to a level of resolution equal to that for whole-genome sequencing. Our assay is designed to be able to accurately subtype the important human-pathogenic species Yersinia enterocolitica to whole-genome resolution levels. We also report the validation of the scheme on 386 strains from reference laboratory collections across Europe. We propose that the scheme is an important molecular typing system to allow accurate and reproducible identification of Yersinia isolates to the species level, a process often inconsistent in nonspecialist laboratories. Additionally, our assay is the most phylogenetically informative typing scheme available for Y. enterocolitica. PMID:25339391

  19. Is multiple-sequence alignment required for accurate inference of phylogeny?

    PubMed

    Höhl, Michael; Ragan, Mark A

    2007-04-01

    the correct phylogeny as accurately as does an approach based on maximum-likelihood distance estimates of multiply aligned sequences.

  20. Case Report: Application of whole exome sequencing for accurate diagnosis of rare syndromes of mineralocorticoid excess

    PubMed Central

    Narayanan, Ranjit; Karuthedath Vellarikkal, Shamsudheen; Jayarajan, Rijith; Verma, Ankit; Dixit, Vishal; Scaria, Vinod; Sivasubbu, Sridhar

    2017-01-01

    Syndromes of mineralocorticoid excess (SME) are closely related clinical manifestations occurring within a specific set of diseases. Overlapping clinical manifestations of such syndromes often create a dilemma in accurate diagnosis, which is crucial for disease surveillance and management especially in rare genetic disorders. Here we demonstrate the use of whole exome sequencing (WES) for accurate diagnosis of rare SME and report that p.R337C variation in the HSD11B2 gene causes progressive apparent mineralocorticoid excess (AME) syndrome in a South Indian family of Mappila origin. PMID:29067160

  1. The identification of complete domains within protein sequences using accurate E-values for semi-global alignment

    PubMed Central

    Kann, Maricel G.; Sheetlin, Sergey L.; Park, Yonil; Bryant, Stephen H.; Spouge, John L.

    2007-01-01

    The sequencing of complete genomes has created a pressing need for automated annotation of gene function. Because domains are the basic units of protein function and evolution, a gene can be annotated from a domain database by aligning domains to the corresponding protein sequence. Ideally, complete domains are aligned to protein subsequences, in a ‘semi-global alignment’. Local alignment, which aligns pieces of domains to subsequences, is common in high-throughput annotation applications, however. It is a mature technique, with the heuristics and accurate E-values required for screening large databases and evaluating the screening results. Hidden Markov models (HMMs) provide an alternative theoretical framework for semi-global alignment, but their use is limited because they lack heuristic acceleration and accurate E-values. Our new tool, GLOBAL, overcomes some limitations of previous semi-global HMMs: it has accurate E-values and the possibility of the heuristic acceleration required for high-throughput applications. Moreover, according to a standard of truth based on protein structure, two semi-global HMM alignment tools (GLOBAL and HMMer) had comparable performance in identifying complete domains, but distinctly outperformed two tools based on local alignment. When searching for complete protein domains, therefore, GLOBAL avoids disadvantages commonly associated with HMMs, yet maintains their superior retrieval performance. PMID:17596268

  2. Accurate typing of short tandem repeats from genome-wide sequencing data and its applications.

    PubMed

    Fungtammasan, Arkarachai; Ananda, Guruprasad; Hile, Suzanne E; Su, Marcia Shu-Wei; Sun, Chen; Harris, Robert; Medvedev, Paul; Eckert, Kristin; Makova, Kateryna D

    2015-05-01

    Short tandem repeats (STRs) are implicated in dozens of human genetic diseases and contribute significantly to genome variation and instability. Yet profiling STRs from short-read sequencing data is challenging because of their high sequencing error rates. Here, we developed STR-FM, short tandem repeat profiling using flank-based mapping, a computational pipeline that can detect the full spectrum of STR alleles from short-read data, can adapt to emerging read-mapping algorithms, and can be applied to heterogeneous genetic samples (e.g., tumors, viruses, and genomes of organelles). We used STR-FM to study STR error rates and patterns in publicly available human and in-house generated ultradeep plasmid sequencing data sets. We discovered that STRs sequenced with a PCR-free protocol have up to ninefold fewer errors than those sequenced with a PCR-containing protocol. We constructed an error correction model for genotyping STRs that can distinguish heterozygous alleles containing STRs with consecutive repeat numbers. Applying our model and pipeline to Illumina sequencing data with 100-bp reads, we could confidently genotype several disease-related long trinucleotide STRs. Utilizing this pipeline, for the first time we determined the genome-wide STR germline mutation rate from a deeply sequenced human pedigree. Additionally, we built a tool that recommends minimal sequencing depth for accurate STR genotyping, depending on repeat length and sequencing read length. The required read depth increases with STR length and is lower for a PCR-free protocol. This suite of tools addresses the pressing challenges surrounding STR genotyping, and thus is of wide interest to researchers investigating disease-related STRs and STR evolution. © 2015 Fungtammasan et al.; Published by Cold Spring Harbor Laboratory Press.

  3. Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer.

    PubMed

    Wang, Ji; Ke, Yue Hua; Zhang, Yong; Huang, Ke Qiang; Wang, Lei; Shen, Xin Xin; Dong, Xiao Ping; Xu, Wen Bo; Ma, Xue Jun

    2017-10-01

    Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run. MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  4. A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA.

    PubMed

    Ludgate, Jackie L; Wright, James; Stockwell, Peter A; Morison, Ian M; Eccles, Michael R; Chatterjee, Aniruddha

    2017-08-31

    Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis. Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing. The main features and advantages of this protocol are: An optimized method for extracting good quality DNA from FFPE tissues. An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue. Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing. We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.

  5. Accurate Typing of Human Leukocyte Antigen Class I Genes by Oxford Nanopore Sequencing.

    PubMed

    Liu, Chang; Xiao, Fangzhou; Hoisington-Lopez, Jessica; Lang, Kathrin; Quenzel, Philipp; Duffy, Brian; Mitra, Robi David

    2018-04-03

    Oxford Nanopore Technologies' MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability. The MinION has the potential to enable expedited point-of-care human leukocyte antigen (HLA) typing, an assay routinely used to assess the immunologic compatibility between organ donors and recipients, but the platform's high error rate makes it challenging to type alleles with accuracy. We developed and validated accurate typing of HLA by Oxford nanopore (Athlon), a bioinformatic pipeline that i) maps nanopore reads to a database of known HLA alleles, ii) identifies candidate alleles with the highest read coverage at different resolution levels that are represented as branching nodes and leaves of a tree structure, iii) generates consensus sequences by remapping the reads to the candidate alleles, and iv) calls the final diploid genotype by blasting consensus sequences against the reference database. Using two independent data sets generated on the R9.4 flow cell chemistry, Athlon achieved a 100% accuracy in class I HLA typing at the two-field resolution. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  6. Accurate estimation of object location in an image sequence using helicopter flight data

    NASA Technical Reports Server (NTRS)

    Tang, Yuan-Liang; Kasturi, Rangachar

    1994-01-01

    In autonomous navigation, it is essential to obtain a three-dimensional (3D) description of the static environment in which the vehicle is traveling. For a rotorcraft conducting low-latitude flight, this description is particularly useful for obstacle detection and avoidance. In this paper, we address the problem of 3D position estimation for static objects from a monocular sequence of images captured from a low-latitude flying helicopter. Since the environment is static, it is well known that the optical flow in the image will produce a radiating pattern from the focus of expansion. We propose a motion analysis system which utilizes the epipolar constraint to accurately estimate 3D positions of scene objects in a real world image sequence taken from a low-altitude flying helicopter. Results show that this approach gives good estimates of object positions near the rotorcraft's intended flight-path.

  7. Consistency of VDJ Rearrangement and Substitution Parameters Enables Accurate B Cell Receptor Sequence Annotation.

    PubMed

    Ralph, Duncan K; Matsen, Frederick A

    2016-01-01

    VDJ rearrangement and somatic hypermutation work together to produce antibody-coding B cell receptor (BCR) sequences for a remarkable diversity of antigens. It is now possible to sequence these BCRs in high throughput; analysis of these sequences is bringing new insight into how antibodies develop, in particular for broadly-neutralizing antibodies against HIV and influenza. A fundamental step in such sequence analysis is to annotate each base as coming from a specific one of the V, D, or J genes, or from an N-addition (a.k.a. non-templated insertion). Previous work has used simple parametric distributions to model transitions from state to state in a hidden Markov model (HMM) of VDJ recombination, and assumed that mutations occur via the same process across sites. However, codon frame and other effects have been observed to violate these parametric assumptions for such coding sequences, suggesting that a non-parametric approach to modeling the recombination process could be useful. In our paper, we find that indeed large modern data sets suggest a model using parameter-rich per-allele categorical distributions for HMM transition probabilities and per-allele-per-position mutation probabilities, and that using such a model for inference leads to significantly improved results. We present an accurate and efficient BCR sequence annotation software package using a novel HMM "factorization" strategy. This package, called partis (https://github.com/psathyrella/partis/), is built on a new general-purpose HMM compiler that can perform efficient inference given a simple text description of an HMM.

  8. Rapid detection, classification and accurate alignment of up to a million or more related protein sequences.

    PubMed

    Neuwald, Andrew F

    2009-08-01

    The patterns of sequence similarity and divergence present within functionally diverse, evolutionarily related proteins contain implicit information about corresponding biochemical similarities and differences. A first step toward accessing such information is to statistically analyze these patterns, which, in turn, requires that one first identify and accurately align a very large set of protein sequences. Ideally, the set should include many distantly related, functionally divergent subgroups. Because it is extremely difficult, if not impossible for fully automated methods to align such sequences correctly, researchers often resort to manual curation based on detailed structural and biochemical information. However, multiply-aligning vast numbers of sequences in this way is clearly impractical. This problem is addressed using Multiply-Aligned Profiles for Global Alignment of Protein Sequences (MAPGAPS). The MAPGAPS program uses a set of multiply-aligned profiles both as a query to detect and classify related sequences and as a template to multiply-align the sequences. It relies on Karlin-Altschul statistics for sensitivity and on PSI-BLAST (and other) heuristics for speed. Using as input a carefully curated multiple-profile alignment for P-loop GTPases, MAPGAPS correctly aligned weakly conserved sequence motifs within 33 distantly related GTPases of known structure. By comparison, the sequence- and structurally based alignment methods hmmalign and PROMALS3D misaligned at least 11 and 23 of these regions, respectively. When applied to a dataset of 65 million protein sequences, MAPGAPS identified, classified and aligned (with comparable accuracy) nearly half a million putative P-loop GTPase sequences. A C++ implementation of MAPGAPS is available at http://mapgaps.igs.umaryland.edu. Supplementary data are available at Bioinformatics online.

  9. 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.

  10. BayMeth: improved DNA methylation quantification for affinity capture sequencing data using a flexible Bayesian approach

    PubMed Central

    2014-01-01

    Affinity capture of DNA methylation combined with high-throughput sequencing strikes a good balance between the high cost of whole genome bisulfite sequencing and the low coverage of methylation arrays. We present BayMeth, an empirical Bayes approach that uses a fully methylated control sample to transform observed read counts into regional methylation levels. In our model, inefficient capture can readily be distinguished from low methylation levels. BayMeth improves on existing methods, allows explicit modeling of copy number variation, and offers computationally efficient analytical mean and variance estimators. BayMeth is available in the Repitools Bioconductor package. PMID:24517713

  11. Single-Cell Sequencing for Drug Discovery and Drug Development.

    PubMed

    Wu, Hongjin; Wang, Charles; Wu, Shixiu

    2017-01-01

    Next-generation sequencing (NGS), particularly single-cell sequencing, has revolutionized the scale and scope of genomic and biomedical research. Recent technological advances in NGS and singlecell studies have made the deep whole-genome (DNA-seq), whole epigenome and whole-transcriptome sequencing (RNA-seq) at single-cell level feasible. NGS at the single-cell level expands our view of genome, epigenome and transcriptome and allows the genome, epigenome and transcriptome of any organism to be explored without a priori assumptions and with unprecedented throughput. And it does so with single-nucleotide resolution. NGS is also a very powerful tool for drug discovery and drug development. In this review, we describe the current state of single-cell sequencing techniques, which can provide a new, more powerful and precise approach for analyzing effects of drugs on treated cells and tissues. Our review discusses single-cell whole genome/exome sequencing (scWGS/scWES), single-cell transcriptome sequencing (scRNA-seq), single-cell bisulfite sequencing (scBS), and multiple omics of single-cell sequencing. We also highlight the advantages and challenges of each of these approaches. Finally, we describe, elaborate and speculate the potential applications of single-cell sequencing for drug discovery and drug development. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Combined Bisulfite Restriction Analysis for brain tissue identification.

    PubMed

    Samsuwan, Jarunya; Muangsub, Tachapol; Yanatatsaneejit, Pattamawadee; Mutirangura, Apiwat; Kitkumthorn, Nakarin

    2018-05-01

    According to the tissue-specific methylation database (doi: 10.1016/j.gene.2014.09.060), methylation at CpG locus cg03096975 in EML2 has been preliminarily proven to be specific to brain tissue. In this study, we enlarged sample size and developed a technique for identifying brain tissue in aged samples. Combined Bisulfite Restriction Analysis-for EML2 (COBRA-EML2) technique was established and validated in various organ samples obtained from 108 autopsies. In addition, this technique was also tested for its reliability, minimal DNA concentration detected, and use in aged samples and in samples obtained from specific brain compartments and spinal cord. COBRA-EML2 displayed 100% sensitivity and specificity for distinguishing brain tissue from other tissues, showed high reliability, was capable of detecting minimal DNA concentration (0.015ng/μl), could be used for identifying brain tissue in aged samples. In summary, COBRA-EML2 is a technique to identify brain tissue. This analysis is useful in criminal cases since it can identify the vital organ tissues from small samples acquired from criminal scenes. The results from this analysis can be counted as a medical and forensic marker supporting criminal investigations, and as one of the evidences in court rulings. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Identification of differentially methylated sites with weak methylation effect

    USDA-ARS?s Scientific Manuscript database

    DNA methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect dif...

  14. CloudAligner: A fast and full-featured MapReduce based tool for sequence mapping.

    PubMed

    Nguyen, Tung; Shi, Weisong; Ruden, Douglas

    2011-06-06

    Research in genetics has developed rapidly recently due to the aid of next generation sequencing (NGS). However, massively-parallel NGS produces enormous amounts of data, which leads to storage, compatibility, scalability, and performance issues. The Cloud Computing and MapReduce framework, which utilizes hundreds or thousands of shared computers to map sequencing reads quickly and efficiently to reference genome sequences, appears to be a very promising solution for these issues. Consequently, it has been adopted by many organizations recently, and the initial results are very promising. However, since these are only initial steps toward this trend, the developed software does not provide adequate primary functions like bisulfite, pair-end mapping, etc., in on-site software such as RMAP or BS Seeker. In addition, existing MapReduce-based applications were not designed to process the long reads produced by the most recent second-generation and third-generation NGS instruments and, therefore, are inefficient. Last, it is difficult for a majority of biologists untrained in programming skills to use these tools because most were developed on Linux with a command line interface. To urge the trend of using Cloud technologies in genomics and prepare for advances in second- and third-generation DNA sequencing, we have built a Hadoop MapReduce-based application, CloudAligner, which achieves higher performance, covers most primary features, is more accurate, and has a user-friendly interface. It was also designed to be able to deal with long sequences. The performance gain of CloudAligner over Cloud-based counterparts (35 to 80%) mainly comes from the omission of the reduce phase. In comparison to local-based approaches, the performance gain of CloudAligner is from the partition and parallel processing of the huge reference genome as well as the reads. The source code of CloudAligner is available at http://cloudaligner.sourceforge.net/ and its web version is at http

  15. Accurate estimation of short read mapping quality for next-generation genome sequencing

    PubMed Central

    Ruffalo, Matthew; Koyutürk, Mehmet; Ray, Soumya; LaFramboise, Thomas

    2012-01-01

    Motivation: Several software tools specialize in the alignment of short next-generation sequencing reads to a reference sequence. Some of these tools report a mapping quality score for each alignment—in principle, this quality score tells researchers the likelihood that the alignment is correct. However, the reported mapping quality often correlates weakly with actual accuracy and the qualities of many mappings are underestimated, encouraging the researchers to discard correct mappings. Further, these low-quality mappings tend to correlate with variations in the genome (both single nucleotide and structural), and such mappings are important in accurately identifying genomic variants. Approach: We develop a machine learning tool, LoQuM (LOgistic regression tool for calibrating the Quality of short read mappings, to assign reliable mapping quality scores to mappings of Illumina reads returned by any alignment tool. LoQuM uses statistics on the read (base quality scores reported by the sequencer) and the alignment (number of matches, mismatches and deletions, mapping quality score returned by the alignment tool, if available, and number of mappings) as features for classification and uses simulated reads to learn a logistic regression model that relates these features to actual mapping quality. Results: We test the predictions of LoQuM on an independent dataset generated by the ART short read simulation software and observe that LoQuM can ‘resurrect’ many mappings that are assigned zero quality scores by the alignment tools and are therefore likely to be discarded by researchers. We also observe that the recalibration of mapping quality scores greatly enhances the precision of called single nucleotide polymorphisms. Availability: LoQuM is available as open source at http://compbio.case.edu/loqum/. Contact: matthew.ruffalo@case.edu. PMID:22962451

  16. Inhibition of the. beta. -carboxylation pathway of CO/sub 2/ fixation by bisulfite compounds. [Leaves of Sedum praealtum and Atriplex spongiosa were used

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

    Osmond, C.B.; Avadhani, P.N.

    1970-01-01

    Bisulfite compounds are well known as inhibitors of glycolate oxidase in green tissues of higher plants. In an effort to understand the relation between low glycolate oxidase activity and high P-enolpyruvate carboxylase activity in plants with the C/sub 4/ dicarboxylic acid pathway of photosynthesis, the authors have treated leaves of related species of Atriplex with these compounds. In this photosynthetic process, as well as during dark CO/sub 2/ fixation leading to acidification of Sedum leaves, they have found bisulfite compounds to be effective inhibitors of the P-enolpyruvate carboxylation system. This report provides evidence in vivo for this inhibition and describesmore » the inhibition in vitro of P-enolpyruvate carboxylation system. This report provides evidence in vivo for this inhibition and describes the inhibition in vitro of P-enolpyruvate carboxylase and NADH malate dehydrogenase. 16 references, 4 figures, 1 table.« less

  17. MethPrimer: designing primers for methylation PCRs.

    PubMed

    Li, Long-Cheng; Dahiya, Rajvir

    2002-11-01

    DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications. MethPrimer, based on Primer 3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.

  18. Non-invasive prenatal diagnosis of achondroplasia and thanatophoric dysplasia: next-generation sequencing allows for a safer, more accurate, and comprehensive approach

    PubMed Central

    Chitty, Lyn S; Mason, Sarah; Barrett, Angela N; McKay, Fiona; Lench, Nicholas; Daley, Rebecca; Jenkins, Lucy A

    2015-01-01

    Abstract Objective Accurate prenatal diagnosis of genetic conditions can be challenging and usually requires invasive testing. Here, we demonstrate the potential of next-generation sequencing (NGS) for the analysis of cell-free DNA in maternal blood to transform prenatal diagnosis of monogenic disorders. Methods Analysis of cell-free DNA using a PCR and restriction enzyme digest (PCR–RED) was compared with a novel NGS assay in pregnancies at risk of achondroplasia and thanatophoric dysplasia. Results PCR–RED was performed in 72 cases and was correct in 88.6%, inconclusive in 7% with one false negative. NGS was performed in 47 cases and was accurate in 96.2% with no inconclusives. Both approaches were used in 27 cases, with NGS giving the correct result in the two cases inconclusive with PCR–RED. Conclusion NGS provides an accurate, flexible approach to non-invasive prenatal diagnosis of de novo and paternally inherited mutations. It is more sensitive than PCR–RED and is ideal when screening a gene with multiple potential pathogenic mutations. These findings highlight the value of NGS in the development of non-invasive prenatal diagnosis for other monogenic disorders. © 2015 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd. What's already known about this topic? Non-invasive prenatal diagnosis (NIPD) using PCR-based methods has been reported for the detection or exclusion of individual paternally inherited or de novo alleles in maternal plasma. What does this study add? NIPD using next generation sequencing provides an accurate, more sensitive approach which can be used to detect multiple mutations in a single assay and so is ideal when screening a gene with multiple potential pathogenic mutations. Next generation sequencing thus provides a flexible approach to non-invasive prenatal diagnosis ideal for use in a busy service laboratory. PMID:25728633

  19. Accurate multiplex polony sequencing of an evolved bacterial genome.

    PubMed

    Shendure, Jay; Porreca, Gregory J; Reppas, Nikos B; Lin, Xiaoxia; McCutcheon, John P; Rosenbaum, Abraham M; Wang, Michael D; Zhang, Kun; Mitra, Robi D; Church, George M

    2005-09-09

    We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

  20. A highly sensitive and accurate gene expression analysis by sequencing ("bead-seq") for a single cell.

    PubMed

    Matsunaga, Hiroko; Goto, Mari; Arikawa, Koji; Shirai, Masataka; Tsunoda, Hiroyuki; Huang, Huan; Kambara, Hideki

    2015-02-15

    Analyses of gene expressions in single cells are important for understanding detailed biological phenomena. Here, a highly sensitive and accurate method by sequencing (called "bead-seq") to obtain a whole gene expression profile for a single cell is proposed. A key feature of the method is to use a complementary DNA (cDNA) library on magnetic beads, which enables adding washing steps to remove residual reagents in a sample preparation process. By adding the washing steps, the next steps can be carried out under the optimal conditions without losing cDNAs. Error sources were carefully evaluated to conclude that the first several steps were the key steps. It is demonstrated that bead-seq is superior to the conventional methods for single-cell gene expression analyses in terms of reproducibility, quantitative accuracy, and biases caused during sample preparation and sequencing processes. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Modeling the Kinetics of Contaminants Oxidation and the Generation of Manganese(III) in the Permanganate/Bisulfite Process.

    PubMed

    Sun, Bo; Dong, Hongyu; He, Di; Rao, Dandan; Guan, Xiaohong

    2016-02-02

    Permanganate can be activated by bisulfite to generate soluble Mn(III) (noncomplexed with ligands other than H2O and OH(-)) which oxidizes organic contaminants at extraordinarily high rates. However, the generation of Mn(III) in the permanganate/bisulfite (PM/BS) process and the reactivity of Mn(III) toward emerging contaminants have never been quantified. In this work, Mn(III) generated in the PM/BS process was shown to absorb at 230-290 nm for the first time and disproportionated more easily at higher pH, and thus, the utilization rate of Mn(III) for decomposing organic contaminant was low under alkaline conditions. A Mn(III) generation and utilization model was developed to get the second-order reaction rate parameters of benzene oxidation by soluble Mn(III), and then, benzene was chosen as the reference probe to build a competition kinetics method, which was employed to obtain the second-order rate constants of organic contaminants oxidation by soluble Mn(III). The results revealed that the second-order rate constants of aniline and bisphenol A oxidation by soluble Mn(III) were in the range of 10(5)-10(6) M(-1) s(-1). With the presence of soluble Mn(III) at micromolar concentration, contaminants could be oxidized with the observed rates several orders of magnitude higher than those by common oxidation processes, implying the great potential application of the PM/BS process in water and wastewater treatment.

  2. Targeted-bisulfite sequence analysis of the methylation of CpG islands in genes encoding PNPLA3, SAMM50, and PARVB of patients with non-alcoholic fatty liver disease.

    PubMed

    Kitamoto, Takuya; Kitamoto, Aya; Ogawa, Yuji; Honda, Yasushi; Imajo, Kento; Saito, Satoru; Yoneda, Masato; Nakamura, Takahiro; Nakajima, Atsushi; Hotta, Kikuko

    2015-08-01

    The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is affected by epigenetic factors as well as by genetic variation. We performed targeted-bisulfite sequencing to determine the levels of DNA methylation of 4 CpG islands (CpG99, CpG71, CpG26, and CpG101) in the regulatory regions of PNPLA3, SAMM50, PARVB variant 1, and PARVB variant 2, respectively. We compared the levels of methylation of DNA in the livers of the first and second sets of patients with mild (fibrosis stages 0 and 1) or advanced (fibrosis stages 2 to 4) NAFLD and in those of patients with mild (F0 to F2) or advanced (F3 and F4) chronic hepatitis C infection. The hepatic mRNA levels of PNPLA3, SAMM50, and PARVB were measured using qPCR. CpG26, which resides in the regulatory region of PARVB variant 1, was markedly hypomethylated in the livers of patients with advanced NAFLD. Conversely, CpG99 in the regulatory region of PNPLA3 was substantially hypermethylated in these patients. These differences in DNA methylation were replicated in a second set of patients with NAFLD or chronic hepatitis C. PNPLA3 mRNA levels in the liver of the same section of a biopsy specimen used for genomic DNA preparation were lower in patients with advanced NAFLD compared with those with mild NAFLD and correlated inversely with CpG99 methylation in liver DNA. Moreover, the levels of CpG99 methylation and PNPLA3 mRNA were affected by the rs738409 genotype. Hypomethylation of CpG26 and hypermethylation of CpG99 may contribute to the severity of fibrosis in patients with NAFLD or chronic hepatitis C infection. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  3. Direct reductive amination of aldehyde bisulfite adducts induced by 2-picoline borane: application to the synthesis of a DPP-IV inhibitor.

    PubMed

    Faul, Margaret; Larsen, Rob; Levinson, Adam; Tedrow, Jason; Vounatsos, Filisaty

    2013-02-15

    Aldehyde-bisulfite adducts dervied from unstable parent aldehydes were reductively alkylated in a direct fashion with a variety of amines. This approach features the use of 2-picoline borane as the reducing agent and a protic solvent for the reaction media and has been successfully applied to the synthesis of a DPP-IV inhibitor and a variety of other amines.

  4. NMRDSP: an accurate prediction of protein shape strings from NMR chemical shifts and sequence data.

    PubMed

    Mao, Wusong; Cong, Peisheng; Wang, Zhiheng; Lu, Longjian; Zhu, Zhongliang; Li, Tonghua

    2013-01-01

    Shape string is structural sequence and is an extremely important structure representation of protein backbone conformations. Nuclear magnetic resonance chemical shifts give a strong correlation with the local protein structure, and are exploited to predict protein structures in conjunction with computational approaches. Here we demonstrate a novel approach, NMRDSP, which can accurately predict the protein shape string based on nuclear magnetic resonance chemical shifts and structural profiles obtained from sequence data. The NMRDSP uses six chemical shifts (HA, H, N, CA, CB and C) and eight elements of structure profiles as features, a non-redundant set (1,003 entries) as the training set, and a conditional random field as a classification algorithm. For an independent testing set (203 entries), we achieved an accuracy of 75.8% for S8 (the eight states accuracy) and 87.8% for S3 (the three states accuracy). This is higher than only using chemical shifts or sequence data, and confirms that the chemical shift and the structure profile are significant features for shape string prediction and their combination prominently improves the accuracy of the predictor. We have constructed the NMRDSP web server and believe it could be employed to provide a solid platform to predict other protein structures and functions. The NMRDSP web server is freely available at http://cal.tongji.edu.cn/NMRDSP/index.jsp.

  5. COPS: A Sensitive and Accurate Tool for Detecting Somatic Copy Number Alterations Using Short-Read Sequence Data from Paired Samples

    PubMed Central

    Krishnan, Neeraja M.; Gaur, Prakhar; Chaudhary, Rakshit; Rao, Arjun A.; Panda, Binay

    2012-01-01

    Copy Number Alterations (CNAs) such as deletions and duplications; compose a larger percentage of genetic variations than single nucleotide polymorphisms or other structural variations in cancer genomes that undergo major chromosomal re-arrangements. It is, therefore, imperative to identify cancer-specific somatic copy number alterations (SCNAs), with respect to matched normal tissue, in order to understand their association with the disease. We have devised an accurate, sensitive, and easy-to-use tool, COPS, COpy number using Paired Samples, for detecting SCNAs. We rigorously tested the performance of COPS using short sequence simulated reads at various sizes and coverage of SCNAs, read depths, read lengths and also with real tumor:normal paired samples. We found COPS to perform better in comparison to other known SCNA detection tools for all evaluated parameters, namely, sensitivity (detection of true positives), specificity (detection of false positives) and size accuracy. COPS performed well for sequencing reads of all lengths when used with most upstream read alignment tools. Additionally, by incorporating a downstream boundary segmentation detection tool, the accuracy of SCNA boundaries was further improved. Here, we report an accurate, sensitive and easy to use tool in detecting cancer-specific SCNAs using short-read sequence data. In addition to cancer, COPS can be used for any disease as long as sequence reads from both disease and normal samples from the same individual are available. An added boundary segmentation detection module makes COPS detected SCNA boundaries more specific for the samples studied. COPS is available at ftp://115.119.160.213 with username “cops” and password “cops”. PMID:23110103

  6. Seshat: A Web service for accurate annotation, validation, and analysis of TP53 variants generated by conventional and next-generation sequencing.

    PubMed

    Tikkanen, Tuomas; Leroy, Bernard; Fournier, Jean Louis; Risques, Rosa Ana; Malcikova, Jitka; Soussi, Thierry

    2018-07-01

    Accurate annotation of genomic variants in human diseases is essential to allow personalized medicine. Assessment of somatic and germline TP53 alterations has now reached the clinic and is required in several circumstances such as the identification of the most effective cancer therapy for patients with chronic lymphocytic leukemia (CLL). Here, we present Seshat, a Web service for annotating TP53 information derived from sequencing data. A flexible framework allows the use of standard file formats such as Mutation Annotation Format (MAF) or Variant Call Format (VCF), as well as common TXT files. Seshat performs accurate variant annotations using the Human Genome Variation Society (HGVS) nomenclature and the stable TP53 genomic reference provided by the Locus Reference Genomic (LRG). In addition, using the 2017 release of the UMD_TP53 database, Seshat provides multiple statistical information for each TP53 variant including database frequency, functional activity, or pathogenicity. The information is delivered in standardized output tables that minimize errors and facilitate comparison of mutational data across studies. Seshat is a beneficial tool to interpret the ever-growing TP53 sequencing data generated by multiple sequencing platforms and it is freely available via the TP53 Website, http://p53.fr or directly at http://vps338341.ovh.net/. © 2018 Wiley Periodicals, Inc.

  7. On-Chip Evaluation of DNA Methylation with Electrochemical Combined Bisulfite Restriction Analysis Utilizing a Carbon Film Containing a Nanocrystalline Structure.

    PubMed

    Kurita, Ryoji; Yanagisawa, Hiroyuki; Kamata, Tomoyuki; Kato, Dai; Niwa, Osamu

    2017-06-06

    This paper reports an on-chip electrochemical assessment of the DNA methylation status in genomic DNA on a conductive nanocarbon film electrode realized with combined bisulfite restriction analysis (COBRA). The film electrode consists of sp 2 and sp 3 hybrid bonds and is fabricated with an unbalanced magnetron (UBM) sputtering method. First, we studied the effect of the sp 2 /sp 3 ratio of the UBM nanocarbon film electrode with p-aminophenol, which is a major electro-active product of the labeling enzyme from p-aminophenol phosphate. The signal current for p-aminophenol increases as the sp 2 content in the UBM nanocarbon film electrode increases because of the π-π interaction between aromatic p-aminophenol and the graphene-like sp 2 structure. Furthermore, the capacitative current at the UBM nanocarbon film electrode was successfully reduced by about 1 order of magnitude thanks to the angstrom-level surface flatness. Therefore, a high signal-to-noise ratio was achieved compared with that of conventional electrodes. Then, after performing an ELISA-like hybridization assay with a restriction enzyme, we undertook an electrochemical evaluation of the cytosine methylation status in DNA by measuring the oxidation current derived from p-aminophenol. When the target cytosine in the analyte sequence is methylated (unmethylated), the restriction enzyme of HpyCH4IV is able (unable) to cleave the sequence, that is, the detection probe cannot (can) hybridize. We succeeded in estimating the methylation ratio at a site-specific CpG site from the peak current of a cyclic voltammogram obtained from a PCR product solution ranging from 0.01 to 1 nM.

  8. DNA hypomethylation of individual sequences in aborted cloned bovine fetuses.

    PubMed

    Chen, Tao; Jiang, Yan; Zhang, Yan-Ling; Liu, Jing-He; Hou, Yi; Schatten, Heide; Chen, Da-Yuan; Sun, Qing-Yuan

    2005-09-01

    Cloned bovines have a much higher abortion rate than those derived in vivo. Available evidence indicates that inappropriate epigenetic reprogramming of donor nuclei is the primary cause of cloning failure. To gain a better understanding of the DNA methylation changes associated with the high abortion rate of cloned bovines, we examined the DNA methylation status of a repeated sequence (satellite I) and the promoter regions of two single-copy genes (interleukin 3/cytokeratin) in aborted cloned fetuses, aborted fetuses derived from artificial insemination (AI), cloned adults and AI adults by bisulfite sequencing and restriction enzyme analysis. Two of four aborted cloned fetuses show very low methylation levels in the two single-copy gene promoter regions. One of the two fetuses also showed undermethylated status in the satellite I sequence. The other two aborted cloned fetuses have similar methylation levels to those of aborted AI fetuses. However, no difference in methylation was observed between cloned adults and AI adults. Our results demonstrate for the first time the undermethylated status of individual sequences in aborted cloned fetuses. These findings suggest that aberrant DNA methylation may contribute to the developmental failure of cloned bovine fetuses.

  9. DNA barcode data accurately assign higher spider taxa

    PubMed Central

    Coddington, Jonathan A.; Agnarsson, Ingi; Cheng, Ren-Chung; Čandek, Klemen; Driskell, Amy; Frick, Holger; Gregorič, Matjaž; Kostanjšek, Rok; Kropf, Christian; Kweskin, Matthew; Lokovšek, Tjaša; Pipan, Miha; Vidergar, Nina

    2016-01-01

    The use of unique DNA sequences as a method for taxonomic identification is no longer fundamentally controversial, even though debate continues on the best markers, methods, and technology to use. Although both existing databanks such as GenBank and BOLD, as well as reference taxonomies, are imperfect, in best case scenarios “barcodes” (whether single or multiple, organelle or nuclear, loci) clearly are an increasingly fast and inexpensive method of identification, especially as compared to manual identification of unknowns by increasingly rare expert taxonomists. Because most species on Earth are undescribed, a complete reference database at the species level is impractical in the near term. The question therefore arises whether unidentified species can, using DNA barcodes, be accurately assigned to more inclusive groups such as genera and families—taxonomic ranks of putatively monophyletic groups for which the global inventory is more complete and stable. We used a carefully chosen test library of CO1 sequences from 49 families, 313 genera, and 816 species of spiders to assess the accuracy of genus and family-level assignment. We used BLAST queries of each sequence against the entire library and got the top ten hits. The percent sequence identity was reported from these hits (PIdent, range 75–100%). Accurate assignment of higher taxa (PIdent above which errors totaled less than 5%) occurred for genera at PIdent values >95 and families at PIdent values ≥ 91, suggesting these as heuristic thresholds for accurate generic and familial identifications in spiders. Accuracy of identification increases with numbers of species/genus and genera/family in the library; above five genera per family and fifteen species per genus all higher taxon assignments were correct. We propose that using percent sequence identity between conventional barcode sequences may be a feasible and reasonably accurate method to identify animals to family/genus. However, the quality of

  10. Accurate and exact CNV identification from targeted high-throughput sequence data.

    PubMed

    Nord, Alex S; Lee, Ming; King, Mary-Claire; Walsh, Tom

    2011-04-12

    Massively parallel sequencing of barcoded DNA samples significantly increases screening efficiency for clinically important genes. Short read aligners are well suited to single nucleotide and indel detection. However, methods for CNV detection from targeted enrichment are lacking. We present a method combining coverage with map information for the identification of deletions and duplications in targeted sequence data. Sequencing data is first scanned for gains and losses using a comparison of normalized coverage data between samples. CNV calls are confirmed by testing for a signature of sequences that span the CNV breakpoint. With our method, CNVs can be identified regardless of whether breakpoints are within regions targeted for sequencing. For CNVs where at least one breakpoint is within targeted sequence, exact CNV breakpoints can be identified. In a test data set of 96 subjects sequenced across ~1 Mb genomic sequence using multiplexing technology, our method detected mutations as small as 31 bp, predicted quantitative copy count, and had a low false-positive rate. Application of this method allows for identification of gains and losses in targeted sequence data, providing comprehensive mutation screening when combined with a short read aligner.

  11. A standardized framework for accurate, high-throughput genotyping of recombinant and non-recombinant viral sequences.

    PubMed

    Alcantara, Luiz Carlos Junior; Cassol, Sharon; Libin, Pieter; Deforche, Koen; Pybus, Oliver G; Van Ranst, Marc; Galvão-Castro, Bernardo; Vandamme, Anne-Mieke; de Oliveira, Tulio

    2009-07-01

    Human immunodeficiency virus type-1 (HIV-1), hepatitis B and C and other rapidly evolving viruses are characterized by extremely high levels of genetic diversity. To facilitate diagnosis and the development of prevention and treatment strategies that efficiently target the diversity of these viruses, and other pathogens such as human T-lymphotropic virus type-1 (HTLV-1), human herpes virus type-8 (HHV8) and human papillomavirus (HPV), we developed a rapid high-throughput-genotyping system. The method involves the alignment of a query sequence with a carefully selected set of pre-defined reference strains, followed by phylogenetic analysis of multiple overlapping segments of the alignment using a sliding window. Each segment of the query sequence is assigned the genotype and sub-genotype of the reference strain with the highest bootstrap (>70%) and bootscanning (>90%) scores. Results from all windows are combined and displayed graphically using color-coded genotypes. The new Virus-Genotyping Tools provide accurate classification of recombinant and non-recombinant viruses and are currently being assessed for their diagnostic utility. They have incorporated into several HIV drug resistance algorithms including the Stanford (http://hivdb.stanford.edu) and two European databases (http://www.umcutrecht.nl/subsite/spread-programme/ and http://www.hivrdb.org.uk/) and have been successfully used to genotype a large number of sequences in these and other databases. The tools are a PHP/JAVA web application and are freely accessible on a number of servers including: http://bioafrica.mrc.ac.za/rega-genotype/html/, http://lasp.cpqgm.fiocruz.br/virus-genotype/html/, http://jose.med.kuleuven.be/genotypetool/html/.

  12. Hi-Plex for Simple, Accurate, and Cost-Effective Amplicon-based Targeted DNA Sequencing.

    PubMed

    Pope, Bernard J; Hammet, Fleur; Nguyen-Dumont, Tu; Park, Daniel J

    2018-01-01

    Hi-Plex is a suite of methods to enable simple, accurate, and cost-effective highly multiplex PCR-based targeted sequencing (Nguyen-Dumont et al., Biotechniques 58:33-36, 2015). At its core is the principle of using gene-specific primers (GSPs) to "seed" (or target) the reaction and universal primers to "drive" the majority of the reaction. In this manner, effects on amplification efficiencies across the target amplicons can, to a large extent, be restricted to early seeding cycles. Product sizes are defined within a relatively narrow range to enable high-specificity size selection, replication uniformity across target sites (including in the context of fragmented input DNA such as that derived from fixed tumor specimens (Nguyen-Dumont et al., Biotechniques 55:69-74, 2013; Nguyen-Dumont et al., Anal Biochem 470:48-51, 2015), and application of high-specificity genetic variant calling algorithms (Pope et al., Source Code Biol Med 9:3, 2014; Park et al., BMC Bioinformatics 17:165, 2016). Hi-Plex offers a streamlined workflow that is suitable for testing large numbers of specimens without the need for automation.

  13. Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

    PubMed Central

    Harris, R. Alan; Wang, Ting; Coarfa, Cristian; Nagarajan, Raman P.; Hong, Chibo; Downey, Sara L.; Johnson, Brett E.; Fouse, Shaun D.; Delaney, Allen; Zhao, Yongjun; Olshen, Adam; Ballinger, Tracy; Zhou, Xin; Forsberg, Kevin J.; Gu, Junchen; Echipare, Lorigail; O’Geen, Henriette; Lister, Ryan; Pelizzola, Mattia; Xi, Yuanxin; Epstein, Charles B.; Bernstein, Bradley E.; Hawkins, R. David; Ren, Bing; Chung, Wen-Yu; Gu, Hongcang; Bock, Christoph; Gnirke, Andreas; Zhang, Michael Q.; Haussler, David; Ecker, Joseph; Li, Wei; Farnham, Peggy J.; Waterland, Robert A.; Meissner, Alexander; Marra, Marco A.; Hirst, Martin; Milosavljevic, Aleksandar; Costello, Joseph F.

    2010-01-01

    Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression. PMID:20852635

  14. 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

  15. SINA: accurate high-throughput multiple sequence alignment of ribosomal RNA genes.

    PubMed

    Pruesse, Elmar; Peplies, Jörg; Glöckner, Frank Oliver

    2012-07-15

    In the analysis of homologous sequences, computation of multiple sequence alignments (MSAs) has become a bottleneck. This is especially troublesome for marker genes like the ribosomal RNA (rRNA) where already millions of sequences are publicly available and individual studies can easily produce hundreds of thousands of new sequences. Methods have been developed to cope with such numbers, but further improvements are needed to meet accuracy requirements. In this study, we present the SILVA Incremental Aligner (SINA) used to align the rRNA gene databases provided by the SILVA ribosomal RNA project. SINA uses a combination of k-mer searching and partial order alignment (POA) to maintain very high alignment accuracy while satisfying high throughput performance demands. SINA was evaluated in comparison with the commonly used high throughput MSA programs PyNAST and mothur. The three BRAliBase III benchmark MSAs could be reproduced with 99.3, 97.6 and 96.1 accuracy. A larger benchmark MSA comprising 38 772 sequences could be reproduced with 98.9 and 99.3% accuracy using reference MSAs comprising 1000 and 5000 sequences. SINA was able to achieve higher accuracy than PyNAST and mothur in all performed benchmarks. Alignment of up to 500 sequences using the latest SILVA SSU/LSU Ref datasets as reference MSA is offered at http://www.arb-silva.de/aligner. This page also links to Linux binaries, user manual and tutorial. SINA is made available under a personal use license.

  16. Droplet Digital™ PCR Next-Generation Sequencing Library QC Assay.

    PubMed

    Heredia, Nicholas J

    2018-01-01

    Digital PCR is a valuable tool to quantify next-generation sequencing (NGS) libraries precisely and accurately. Accurately quantifying NGS libraries enable accurate loading of the libraries on to the sequencer and thus improve sequencing performance by reducing under and overloading error. Accurate quantification also benefits users by enabling uniform loading of indexed/barcoded libraries which in turn greatly improves sequencing uniformity of the indexed/barcoded samples. The advantages gained by employing the Droplet Digital PCR (ddPCR™) library QC assay includes the precise and accurate quantification in addition to size quality assessment, enabling users to QC their sequencing libraries with confidence.

  17. Effects of sodium meta bisulfite on diffusion fermentation of fodder beets for fuel ethanol production. [Saccharomyces cerevisiae

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

    Gibbons, W.R.; Westby, C.A.

    1987-01-01

    The authors designed and tested a new process for converting fodder beets to ethanol: continuous diffusion-fermentation. This process utilizes the simultaneous diffusion-fermentation concept of the EX-FERM design; however, it overcomes the material handling problems inherent in that system by utilizing a counterflow tubular auger system. This process also eliminates the need for roller mills or presses and dryers which are required for alcohol recovery from solid phase fermentation. The latter is the only other currently feasible procedure for producing distillably worthwhile amounts of ethanol from fodder beets, sweet sorghum, and other similar feedstocks. Results on the use of sodium metamore » bisulfite (SMB) for contamination control with fermenting fodder beet cubes are reported.« less

  18. Comprehensive Analysis of DNA Methylation Data with RnBeads

    PubMed Central

    Walter, Jörn; Lengauer, Thomas; Bock, Christoph

    2014-01-01

    RnBeads is a software tool for large-scale analysis and interpretation of DNA methylation data, providing a user-friendly analysis workflow that yields detailed hypertext reports (http://rnbeads.mpi-inf.mpg.de). Supported assays include whole genome bisulfite sequencing, reduced representation bisulfite sequencing, Infinium microarrays, and any other protocol that produces high-resolution DNA methylation data. Important applications of RnBeads include the analysis of epigenome-wide association studies and epigenetic biomarker discovery in cancer cohorts. PMID:25262207

  19. CpG PatternFinder: a Windows-based utility program for easy and rapid identification of the CpG methylation status of DNA.

    PubMed

    Xu, Yi-Hua; Manoharan, Herbert T; Pitot, Henry C

    2007-09-01

    The bisulfite genomic sequencing technique is one of the most widely used techniques to study sequence-specific DNA methylation because of its unambiguous ability to reveal DNA methylation status to the order of a single nucleotide. One characteristic feature of the bisulfite genomic sequencing technique is that a number of sample sequence files will be produced from a single DNA sample. The PCR products of bisulfite-treated DNA samples cannot be sequenced directly because they are heterogeneous in nature; therefore they should be cloned into suitable plasmids and then sequenced. This procedure generates an enormous number of sample DNA sequence files as well as adding extra bases belonging to the plasmids to the sequence, which will cause problems in the final sequence comparison. Finding the methylation status for each CpG in each sample sequence is not an easy job. As a result CpG PatternFinder was developed for this purpose. The main functions of the CpG PatternFinder are: (i) to analyze the reference sequence to obtain CpG and non-CpG-C residue position information. (ii) To tailor sample sequence files (delete insertions and mark deletions from the sample sequence files) based on a configuration of ClustalW multiple alignment. (iii) To align sample sequence files with a reference file to obtain bisulfite conversion efficiency and CpG methylation status. And, (iv) to produce graphics, highlighted aligned sequence text and a summary report which can be easily exported to Microsoft Office suite. CpG PatternFinder is designed to operate cooperatively with BioEdit, a freeware on the internet. It can handle up to 100 files of sample DNA sequences simultaneously, and the total CpG pattern analysis process can be finished in minutes. CpG PatternFinder is an ideal software tool for DNA methylation studies to determine the differential methylation pattern in a large number of individuals in a population. Previously we developed the CpG Analyzer program; CpG Pattern

  20. QNB: differential RNA methylation analysis for count-based small-sample sequencing data with a quad-negative binomial model.

    PubMed

    Liu, Lian; Zhang, Shao-Wu; Huang, Yufei; Meng, Jia

    2017-08-31

    As a newly emerged research area, RNA epigenetics has drawn increasing attention recently for the participation of RNA methylation and other modifications in a number of crucial biological processes. Thanks to high throughput sequencing techniques, such as, MeRIP-Seq, transcriptome-wide RNA methylation profile is now available in the form of count-based data, with which it is often of interests to study the dynamics at epitranscriptomic layer. However, the sample size of RNA methylation experiment is usually very small due to its costs; and additionally, there usually exist a large number of genes whose methylation level cannot be accurately estimated due to their low expression level, making differential RNA methylation analysis a difficult task. We present QNB, a statistical approach for differential RNA methylation analysis with count-based small-sample sequencing data. Compared with previous approaches such as DRME model based on a statistical test covering the IP samples only with 2 negative binomial distributions, QNB is based on 4 independent negative binomial distributions with their variances and means linked by local regressions, and in the way, the input control samples are also properly taken care of. In addition, different from DRME approach, which relies only the input control sample only for estimating the background, QNB uses a more robust estimator for gene expression by combining information from both input and IP samples, which could largely improve the testing performance for very lowly expressed genes. QNB showed improved performance on both simulated and real MeRIP-Seq datasets when compared with competing algorithms. And the QNB model is also applicable to other datasets related RNA modifications, including but not limited to RNA bisulfite sequencing, m 1 A-Seq, Par-CLIP, RIP-Seq, etc.

  1. Defiant: (DMRs: easy, fast, identification and ANnoTation) identifies differentially Methylated regions from iron-deficient rat hippocampus.

    PubMed

    Condon, David E; Tran, Phu V; Lien, Yu-Chin; Schug, Jonathan; Georgieff, Michael K; Simmons, Rebecca A; Won, Kyoung-Jae

    2018-02-05

    Identification of differentially methylated regions (DMRs) is the initial step towards the study of DNA methylation-mediated gene regulation. Previous approaches to call DMRs suffer from false prediction, use extreme resources, and/or require library installation and input conversion. We developed a new approach called Defiant to identify DMRs. Employing Weighted Welch Expansion (WWE), Defiant showed superior performance to other predictors in the series of benchmarking tests on artificial and real data. Defiant was subsequently used to investigate DNA methylation changes in iron-deficient rat hippocampus. Defiant identified DMRs close to genes associated with neuronal development and plasticity, which were not identified by its competitor. Importantly, Defiant runs between 5 to 479 times faster than currently available software packages. Also, Defiant accepts 10 different input formats widely used for DNA methylation data. Defiant effectively identifies DMRs for whole-genome bisulfite sequencing (WGBS), reduced-representation bisulfite sequencing (RRBS), Tet-assisted bisulfite sequencing (TAB-seq), and HpaII tiny fragment enrichment by ligation-mediated PCR-tag (HELP) assays.

  2. An Integrated Tool to Study MHC Region: Accurate SNV Detection and HLA Genes Typing in Human MHC Region Using Targeted High-Throughput Sequencing

    PubMed Central

    Liu, Xiao; Xu, Yinyin; Liang, Dequan; Gao, Peng; Sun, Yepeng; Gifford, Benjamin; D’Ascenzo, Mark; Liu, Xiaomin; Tellier, Laurent C. A. M.; Yang, Fang; Tong, Xin; Chen, Dan; Zheng, Jing; Li, Weiyang; Richmond, Todd; Xu, Xun; Wang, Jun; Li, Yingrui

    2013-01-01

    The major histocompatibility complex (MHC) is one of the most variable and gene-dense regions of the human genome. Most studies of the MHC, and associated regions, focus on minor variants and HLA typing, many of which have been demonstrated to be associated with human disease susceptibility and metabolic pathways. However, the detection of variants in the MHC region, and diagnostic HLA typing, still lacks a coherent, standardized, cost effective and high coverage protocol of clinical quality and reliability. In this paper, we presented such a method for the accurate detection of minor variants and HLA types in the human MHC region, using high-throughput, high-coverage sequencing of target regions. A probe set was designed to template upon the 8 annotated human MHC haplotypes, and to encompass the 5 megabases (Mb) of the extended MHC region. We deployed our probes upon three, genetically diverse human samples for probe set evaluation, and sequencing data show that ∼97% of the MHC region, and over 99% of the genes in MHC region, are covered with sufficient depth and good evenness. 98% of genotypes called by this capture sequencing prove consistent with established HapMap genotypes. We have concurrently developed a one-step pipeline for calling any HLA type referenced in the IMGT/HLA database from this target capture sequencing data, which shows over 96% typing accuracy when deployed at 4 digital resolution. This cost-effective and highly accurate approach for variant detection and HLA typing in the MHC region may lend further insight into immune-mediated diseases studies, and may find clinical utility in transplantation medicine research. This one-step pipeline is released for general evaluation and use by the scientific community. PMID:23894464

  3. Learning a weighted sequence model of the nucleosome core and linker yields more accurate predictions in Saccharomyces cerevisiae and Homo sapiens.

    PubMed

    Reynolds, Sheila M; Bilmes, Jeff A; Noble, William Stafford

    2010-07-08

    DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence-301 base pairs, centered at the position to be scored-with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the bulk of the

  4. PASTA: Ultra-Large Multiple Sequence Alignment for Nucleotide and Amino-Acid Sequences.

    PubMed

    Mirarab, Siavash; Nguyen, Nam; Guo, Sheng; Wang, Li-San; Kim, Junhyong; Warnow, Tandy

    2015-05-01

    We introduce PASTA, a new multiple sequence alignment algorithm. PASTA uses a new technique to produce an alignment given a guide tree that enables it to be both highly scalable and very accurate. We present a study on biological and simulated data with up to 200,000 sequences, showing that PASTA produces highly accurate alignments, improving on the accuracy and scalability of the leading alignment methods (including SATé). We also show that trees estimated on PASTA alignments are highly accurate--slightly better than SATé trees, but with substantial improvements relative to other methods. Finally, PASTA is faster than SATé, highly parallelizable, and requires relatively little memory.

  5. Identification of S-3-(hexanal)-glutathione and its bisulfite adduct in grape juice from Vitis vinifera L. cv. Sauvignon blanc as new potential precursors of 3SH.

    PubMed

    Thibon, Cécile; Böcker, Caroline; Shinkaruk, Svitlana; Moine, Virginie; Darriet, Philippe; Dubourdieu, Denis

    2016-05-15

    Two main precursors (S-3-(hexan-1-ol)-l-cysteine and S-3-(hexan-1-ol)-l-glutathione) of 3-sulfanylhexanol (3SH, formerly named 3-mercaptohexanol) have been identified so far in grape juice but a correlation between precursor concentrations in grape juices and 3SH concentrations in wines is not always observed. This suggests that there may be other compounds associated with the aromatic potential. In this work, S-3-(hexanal)-glutathione (Glut-3SH-Al) and its bisulfite (Glut-3SH-SO3) adduct were identified in Sauvignon blanc grape juice by liquid chromatography coupled to Fourier transform mass spectrometry experiments. A partial purification of the compounds was carried out by Medium Pressure Liquid Chromatography (MPLC) on the reverse phase using 5L of grape juice. The addition of synthetized Glut-3SH-Al and Glut-3SH-SO3 in the synthetic medium induced a significant release of 3SH after fermentation. Moreover, we demonstrate that Glut-3SH-Al and its bisulfite adduct are present in grape juice and could be considered as new direct 3SH precursors with molar conversion yields close to 0.4%. Copyright © 2016. Published by Elsevier Ltd.

  6. Accurate and High-Coverage Immune Repertoire Sequencing Reveals Characteristics of Antibody Repertoire Diversification in Young Children with Malaria

    NASA Astrophysics Data System (ADS)

    Jiang, Ning

    Accurately measuring the immune repertoire sequence composition, diversity, and abundance is important in studying repertoire response in infections, vaccinations, and cancer immunology. Using molecular identifiers (MIDs) to tag mRNA molecules is an effective method in improving the accuracy of immune repertoire sequencing (IR-seq). However, it is still difficult to use IR-seq on small amount of clinical samples to achieve a high coverage of the repertoire diversities. This is especially challenging in studying infections and vaccinations where B cell subpopulations with fewer cells, such as memory B cells or plasmablasts, are often of great interest to study somatic mutation patterns and diversity changes. Here, we describe an approach of IR-seq based on the use of MIDs in combination with a clustering method that can reveal more than 80% of the antibody diversity in a sample and can be applied to as few as 1,000 B cells. We applied this to study the antibody repertoires of young children before and during an acute malaria infection. We discovered unexpectedly high levels of somatic hypermutation (SHM) in infants and revealed characteristics of antibody repertoire development in young children that would have a profound impact on immunization in children.

  7. Non-invasive prenatal diagnosis of achondroplasia and thanatophoric dysplasia: next-generation sequencing allows for a safer, more accurate, and comprehensive approach.

    PubMed

    Chitty, Lyn S; Mason, Sarah; Barrett, Angela N; McKay, Fiona; Lench, Nicholas; Daley, Rebecca; Jenkins, Lucy A

    2015-07-01

    Accurate prenatal diagnosis of genetic conditions can be challenging and usually requires invasive testing. Here, we demonstrate the potential of next-generation sequencing (NGS) for the analysis of cell-free DNA in maternal blood to transform prenatal diagnosis of monogenic disorders. Analysis of cell-free DNA using a PCR and restriction enzyme digest (PCR-RED) was compared with a novel NGS assay in pregnancies at risk of achondroplasia and thanatophoric dysplasia. PCR-RED was performed in 72 cases and was correct in 88.6%, inconclusive in 7% with one false negative. NGS was performed in 47 cases and was accurate in 96.2% with no inconclusives. Both approaches were used in 27 cases, with NGS giving the correct result in the two cases inconclusive with PCR-RED. NGS provides an accurate, flexible approach to non-invasive prenatal diagnosis of de novo and paternally inherited mutations. It is more sensitive than PCR-RED and is ideal when screening a gene with multiple potential pathogenic mutations. These findings highlight the value of NGS in the development of non-invasive prenatal diagnosis for other monogenic disorders. © 2015 John Wiley & Sons, Ltd.

  8. An epigenetic aging clock for dogs and wolves.

    PubMed

    Thompson, Michael J; vonHoldt, Bridgett; Horvath, Steve; Pellegrini, Matteo

    2017-03-28

    Several articles describe highly accurate age estimation methods based on human DNA-methylation data. It is not yet known whether similar epigenetic aging clocks can be developed based on blood methylation data from canids. Using Reduced Representation Bisulfite Sequencing, we assessed blood DNA-methylation data from 46 domesticated dogs ( Canis familiaris ) and 62 wild gray wolves ( C. lupus ). By regressing chronological dog age on the resulting CpGs, we defined highly accurate multivariate age estimators for dogs (based on 41 CpGs), wolves (67 CpGs), and both combined (115 CpGs). Age related DNA methylation changes in canids implicate similar gene ontology categories as those observed in humans suggesting an evolutionarily conserved mechanism underlying age-related DNA methylation in mammals.

  9. An epigenetic aging clock for dogs and wolves

    PubMed Central

    Thompson, Michael J.; vonHoldt, Bridgett; Horvath, Steve; Pellegrini, Matteo

    2017-01-01

    Several articles describe highly accurate age estimation methods based on human DNA-methylation data. It is not yet known whether similar epigenetic aging clocks can be developed based on blood methylation data from canids. Using Reduced Representation Bisulfite Sequencing, we assessed blood DNA-methylation data from 46 domesticated dogs (Canis familiaris) and 62 wild gray wolves (C. lupus). By regressing chronological dog age on the resulting CpGs, we defined highly accurate multivariate age estimators for dogs (based on 41 CpGs), wolves (67 CpGs), and both combined (115 CpGs). Age related DNA methylation changes in canids implicate similar gene ontology categories as those observed in humans suggesting an evolutionarily conserved mechanism underlying age-related DNA methylation in mammals. PMID:28373601

  10. Determination of formaldehyde by HPLC as the DNPH derivative following high-volume air sampling onto bisulfite-coated cellulose filters

    NASA Astrophysics Data System (ADS)

    de Andrade, Jailson B.; Tanner, Roger L.

    A method is described for the specific collection of formaldehyde as hydroxymethanesulfonate on bisulfate-coated cellulose filters. Following extraction in aqueous acid and removal on unreacted bisulfite, the hydroxymethanesulfonate is decomposed by base, and HCHO is determined by DNPH (2,4-dinitrophenylhydrazine) derivatization and HPLC. Since the collection efficiency for formaldehyde is moderately high even when sampling ambient air at high-volume flow rates, a limit of detection of 0.2 ppbv is achieved with 30 min sampling times. Interference from acetaldehyde co-collected as 1-hydroxyethanesulfonate is <5% using this procedure. The technique shows promise for both short-term airborne sampling, and as a means of collecting mg-sized samples of HCHO on an inorganic matrix for carbon isotopic analyses.

  11. Accurate phylogenetic classification of DNA fragments based onsequence composition

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

    McHardy, Alice C.; Garcia Martin, Hector; Tsirigos, Aristotelis

    2006-05-01

    Metagenome studies have retrieved vast amounts of sequenceout of a variety of environments, leading to novel discoveries and greatinsights into the uncultured microbial world. Except for very simplecommunities, diversity makes sequence assembly and analysis a verychallenging problem. To understand the structure a 5 nd function ofmicrobial communities, a taxonomic characterization of the obtainedsequence fragments is highly desirable, yet currently limited mostly tothose sequences that contain phylogenetic marker genes. We show that forclades at the rank of domain down to genus, sequence composition allowsthe very accurate phylogenetic 10 characterization of genomic sequence.We developed a composition-based classifier, PhyloPythia, for de novophylogenetic sequencemore » characterization and have trained it on adata setof 340 genomes. By extensive evaluation experiments we show that themethodis accurate across all taxonomic ranks considered, even forsequences that originate fromnovel organisms and are as short as 1kb.Application to two metagenome datasets 15 obtained from samples ofphosphorus-removing sludge showed that the method allows the accurateclassification at genus level of most sequence fragments from thedominant populations, while at the same time correctly characterizingeven larger parts of the samples at higher taxonomic levels.« less

  12. Mapping DNA methylation by transverse current sequencing: Reduction of noise from neighboring nucleotides

    NASA Astrophysics Data System (ADS)

    Alvarez, Jose; Massey, Steven; Kalitsov, Alan; Velev, Julian

    Nanopore sequencing via transverse current has emerged as a competitive candidate for mapping DNA methylation without needed bisulfite-treatment, fluorescent tag, or PCR amplification. By eliminating the error producing amplification step, long read lengths become feasible, which greatly simplifies the assembly process and reduces the time and the cost inherent in current technologies. However, due to the large error rates of nanopore sequencing, single base resolution has not been reached. A very important source of noise is the intrinsic structural noise in the electric signature of the nucleotide arising from the influence of neighboring nucleotides. In this work we perform calculations of the tunneling current through DNA molecules in nanopores using the non-equilibrium electron transport method within an effective multi-orbital tight-binding model derived from first-principles calculations. We develop a base-calling algorithm accounting for the correlations of the current through neighboring bases, which in principle can reduce the error rate below any desired precision. Using this method we show that we can clearly distinguish DNA methylation and other base modifications based on the reading of the tunneling current.

  13. Single-cell DNA methylome sequencing and bioinformatic inference of epigenomic cell-state dynamics.

    PubMed

    Farlik, Matthias; Sheffield, Nathan C; Nuzzo, Angelo; Datlinger, Paul; Schönegger, Andreas; Klughammer, Johanna; Bock, Christoph

    2015-03-03

    Methods for single-cell genome and transcriptome sequencing have contributed to our understanding of cellular heterogeneity, whereas methods for single-cell epigenomics are much less established. Here, we describe a whole-genome bisulfite sequencing (WGBS) assay that enables DNA methylation mapping in very small cell populations (μWGBS) and single cells (scWGBS). Our assay is optimized for profiling many samples at low coverage, and we describe a bioinformatic method that analyzes collections of single-cell methylomes to infer cell-state dynamics. Using these technological advances, we studied epigenomic cell-state dynamics in three in vitro models of cellular differentiation and pluripotency, where we observed characteristic patterns of epigenome remodeling and cell-to-cell heterogeneity. The described method enables single-cell analysis of DNA methylation in a broad range of biological systems, including embryonic development, stem cell differentiation, and cancer. It can also be used to establish composite methylomes that account for cell-to-cell heterogeneity in complex tissue samples. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Rapid and accurate pyrosequencing of angiosperm plastid genomes

    PubMed Central

    Moore, Michael J; Dhingra, Amit; Soltis, Pamela S; Shaw, Regina; Farmerie, William G; Folta, Kevin M; Soltis, Douglas E

    2006-01-01

    Background Plastid genome sequence information is vital to several disciplines in plant biology, including phylogenetics and molecular biology. The past five years have witnessed a dramatic increase in the number of completely sequenced plastid genomes, fuelled largely by advances in conventional Sanger sequencing technology. Here we report a further significant reduction in time and cost for plastid genome sequencing through the successful use of a newly available pyrosequencing platform, the Genome Sequencer 20 (GS 20) System (454 Life Sciences Corporation), to rapidly and accurately sequence the whole plastid genomes of the basal eudicot angiosperms Nandina domestica (Berberidaceae) and Platanus occidentalis (Platanaceae). Results More than 99.75% of each plastid genome was simultaneously obtained during two GS 20 sequence runs, to an average depth of coverage of 24.6× in Nandina and 17.3× in Platanus. The Nandina and Platanus plastid genomes shared essentially identical gene complements and possessed the typical angiosperm plastid structure and gene arrangement. To assess the accuracy of the GS 20 sequence, over 45 kilobases of sequence were generated for each genome using conventional sequencing. Overall error rates of 0.043% and 0.031% were observed in GS 20 sequence for Nandina and Platanus, respectively. More than 97% of all observed errors were associated with homopolymer runs, with ~60% of all errors associated with homopolymer runs of 5 or more nucleotides and ~50% of all errors associated with regions of extensive homopolymer runs. No substitution errors were present in either genome. Error rates were generally higher in the single-copy and noncoding regions of both plastid genomes relative to the inverted repeat and coding regions. Conclusion Highly accurate and essentially complete sequence information was obtained for the Nandina and Platanus plastid genomes using the GS 20 System. More importantly, the high accuracy observed in the GS 20 plastid

  15. [Genome-scale sequence data processing and epigenetic analysis of DNA methylation].

    PubMed

    Wang, Ting-Zhang; Shan, Gao; Xu, Jian-Hong; Xue, Qing-Zhong

    2013-06-01

    A new approach recently developed for detecting cytosine DNA methylation (mC) and analyzing the genome-scale DNA methylation profiling, is called BS-Seq which is based on bisulfite conversion of genomic DNA combined with next-generation sequencing. The method can not only provide an insight into the difference of genome-scale DNA methylation among different organisms, but also reveal the conservation of DNA methylation in all contexts and nucleotide preference for different genomic regions, including genes, exons, and repetitive DNA sequences. It will be helpful to under-stand the epigenetic impacts of cytosine DNA methylation on the regulation of gene expression and maintaining silence of repetitive sequences, such as transposable elements. In this paper, we introduce the preprocessing steps of DNA methylation data, by which cytosine (C) and guanine (G) in the reference sequence are transferred to thymine (T) and adenine (A), and cytosine in reads is transferred to thymine, respectively. We also comprehensively review the main content of the DNA methylation analysis on the genomic scale: (1) the cytosine methylation under the context of different sequences; (2) the distribution of genomic methylcytosine; (3) DNA methylation context and the preference for the nucleotides; (4) DNA- protein interaction sites of DNA methylation; (5) degree of methylation of cytosine in the different structural elements of genes. DNA methylation analysis technique provides a powerful tool for the epigenome study in human and other species, and genes and environment interaction, and founds the theoretical basis for further development of disease diagnostics and therapeutics in human.

  16. Lung Sliding Identification Is Less Accurate in the Left Hemithorax.

    PubMed

    Piette, Eric; Daoust, Raoul; Lambert, Jean; Denault, André

    2017-02-01

    The aim of our study was to compare the accuracy of lung sliding identification for the left and right hemithoraxes, using prerecorded short US sequences, in a group of physicians with mixed clinical and US training. A total of 140 US sequences of a complete respiratory cycle were recorded in the operating room. Each sequence was divided in two, yielding 140 sequences of present lung sliding and 140 sequences of absent lung sliding. Of these 280 sequences, 40 were randomly repeated to assess intraobserver variability, for a total of 320 sequences. Descriptive data, the mean accuracy of each participant, as well as the rate of correct answers for each of the original 280 sequences were tabulated and compared for different subgroups of clinical and US training. A video with examples of present and absent lung sliding and a lung pulse was shown before testing. Two sessions were planned to facilitate the participation of 75 clinicians. In the first group, the rate of accurate lung sliding identification was lower in the left hemithorax than in the right (67.0% [interquartile range (IQR), 43.0-83.0] versus 80.0% [IQR, 57.0-95.0]; P < .001). In the second group, the rate of accurate lung sliding identification was also lower in the left hemithorax than in the right (76.3% [IQR, 42.9-90.9] versus 88.7% [IQR, 63.1-96.9]; P = .001). Mean accuracy rates were 67.5% (95% confidence interval, 65.7-69.4) in the first group and 73.1% (95% confidence interval, 70.7-75.5) in the second (P < .001). Lung sliding identification seems less accurate in the left hemithorax when using a short US examination. This study was done on recorded US sequences and should be repeated in a live clinical situation to confirm our results. © 2016 by the American Institute of Ultrasound in Medicine.

  17. Identification of Differentially Methylated Sites with Weak Methylation Effects

    PubMed Central

    Tran, Hong; Zhu, Hongxiao; Wu, Xiaowei; Kim, Gunjune; Clarke, Christopher R.; Larose, Hailey; Haak, David C.; Westwood, James H.; Zhang, Liqing

    2018-01-01

    Deoxyribonucleic acid (DNA) methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs) among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM) was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ) twins who have different pain sensitivities—both datasets have weak methylation effects of <1%—show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs) are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same concepts, with

  18. Sodium bisulfite improves rhizome yield and quality in Paris polyphylla.

    PubMed

    Yu, Kun; Wang, Yan; Wei, Jian-Rong; Ma, Qing; Wang, Bu-Qiong; Yang, Chang-Hong; Wang, Ming-Hui; Yu, Dan; Li, Jia-Ru

    2010-03-01

    Rhizomes of the perennial herb Paris polyphylla have been used in traditional Chinese medicine for hundreds of years. Agricultural production of the rhizomes requires 7-10 years, which is too long to meet the demand of the medicinal industry. Therefore, studies on improving the yield of the herb and shortening the culturing period are imperative. The present work aimed to investigate the effect of sodium bisulfite (NaHSO (3)) on rhizome yield and quality, as well as some related metabolic features of P. polyphylla. The rhizome yield was improved by NaHSO (3) treatment in long-term experiments conducted during 2006 and 2007, with 2 mM NaHSO (3) giving the highest yield. HPLC analysis revealed that NaHSO (3) treatment increased the total saponin content (49 %), including three pennogenin glycosides and two diosgenin glycosides. In a short-term experiment, NaHSO (3) treatment resulted in an enhanced net photosynthetic rate (Pn) for about 4 days without significant changes in the chlorophyll or carotenoid content. The total soluble sugars and sucrose contents in the leaves also significantly increased after 2 mM NaHSO (3) treatment, whereas the starch content changed only slightly. The activities of the enzymes involved in ammonium assimilation (glutamine synthetase [GS] and glutamate dehydrogenase [GDH]) were not significantly influenced. In a long-term experiment, chlorophylls and carotenoids were not significantly affected, and neither was the starch content in leaves, but the total soluble sugars and sucrose contents in leaves increased significantly. The NaHSO (3) treatment significantly increased GS and GDH activities. These results indicate that NaHSO (3) treatment improved the rhizome yield in P. polyphylla, not only through enhancement of Pn but also by improving carbohydrate accumulation and ammonium assimilation. The increased saponin content after NaHSO (3) treatment was indicative of high rhizome quality. (c) Georg Thieme Verlag KG Stuttgart . New York.

  19. Integrating alignment-based and alignment-free sequence similarity measures for biological sequence classification.

    PubMed

    Borozan, Ivan; Watt, Stuart; Ferretti, Vincent

    2015-05-01

    Alignment-based sequence similarity searches, while accurate for some type of sequences, can produce incorrect results when used on more divergent but functionally related sequences that have undergone the sequence rearrangements observed in many bacterial and viral genomes. Here, we propose a classification model that exploits the complementary nature of alignment-based and alignment-free similarity measures with the aim to improve the accuracy with which DNA and protein sequences are characterized. Our model classifies sequences using a combined sequence similarity score calculated by adaptively weighting the contribution of different sequence similarity measures. Weights are determined independently for each sequence in the test set and reflect the discriminatory ability of individual similarity measures in the training set. Because the similarity between some sequences is determined more accurately with one type of measure rather than another, our classifier allows different sets of weights to be associated with different sequences. Using five different similarity measures, we show that our model significantly improves the classification accuracy over the current composition- and alignment-based models, when predicting the taxonomic lineage for both short viral sequence fragments and complete viral sequences. We also show that our model can be used effectively for the classification of reads from a real metagenome dataset as well as protein sequences. All the datasets and the code used in this study are freely available at https://collaborators.oicr.on.ca/vferretti/borozan_csss/csss.html. ivan.borozan@gmail.com Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

  20. Integrating alignment-based and alignment-free sequence similarity measures for biological sequence classification

    PubMed Central

    Borozan, Ivan; Watt, Stuart; Ferretti, Vincent

    2015-01-01

    Motivation: Alignment-based sequence similarity searches, while accurate for some type of sequences, can produce incorrect results when used on more divergent but functionally related sequences that have undergone the sequence rearrangements observed in many bacterial and viral genomes. Here, we propose a classification model that exploits the complementary nature of alignment-based and alignment-free similarity measures with the aim to improve the accuracy with which DNA and protein sequences are characterized. Results: Our model classifies sequences using a combined sequence similarity score calculated by adaptively weighting the contribution of different sequence similarity measures. Weights are determined independently for each sequence in the test set and reflect the discriminatory ability of individual similarity measures in the training set. Because the similarity between some sequences is determined more accurately with one type of measure rather than another, our classifier allows different sets of weights to be associated with different sequences. Using five different similarity measures, we show that our model significantly improves the classification accuracy over the current composition- and alignment-based models, when predicting the taxonomic lineage for both short viral sequence fragments and complete viral sequences. We also show that our model can be used effectively for the classification of reads from a real metagenome dataset as well as protein sequences. Availability and implementation: All the datasets and the code used in this study are freely available at https://collaborators.oicr.on.ca/vferretti/borozan_csss/csss.html. Contact: ivan.borozan@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25573913

  1. MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

    PubMed

    McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

    2016-06-01

    Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

  2. The limits of protein sequence comparison?

    PubMed Central

    Pearson, William R; Sierk, Michael L

    2010-01-01

    Modern sequence alignment algorithms are used routinely to identify homologous proteins, proteins that share a common ancestor. Homologous proteins always share similar structures and often have similar functions. Over the past 20 years, sequence comparison has become both more sensitive, largely because of profile-based methods, and more reliable, because of more accurate statistical estimates. As sequence and structure databases become larger, and comparison methods become more powerful, reliable statistical estimates will become even more important for distinguishing similarities that are due to homology from those that are due to analogy (convergence). The newest sequence alignment methods are more sensitive than older methods, but more accurate statistical estimates are needed for their full power to be realized. PMID:15919194

  3. Efficient and Accurate Algorithm for Cleaved Fragments Prediction (CFPA) in Protein Sequences Dataset Based on Consensus and Its Variants: A Novel Degradomics Prediction Application.

    PubMed

    El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges; Hajj, Hazem; Kobeissy, Firas H

    2017-01-01

    Degradomics is a novel discipline that involves determination of the proteases/substrate fragmentation profile, called the substrate degradome, and has been recently applied in different disciplines. A major application of degradomics is its utility in the field of biomarkers where the breakdown products (BDPs) of different protease have been investigated. Among the major proteases assessed, calpain and caspase proteases have been associated with the execution phases of the pro-apoptotic and pro-necrotic cell death, generating caspase/calpain-specific cleaved fragments. The distinction between calpain and caspase protein fragments has been applied to distinguish injury mechanisms. Advanced proteomics technology has been used to identify these BDPs experimentally. However, it has been a challenge to identify these BDPs with high precision and efficiency, especially if we are targeting a number of proteins at one time. In this chapter, we present a novel bioinfromatic detection method that identifies BDPs accurately and efficiently with validation against experimental data. This method aims at predicting the consensus sequence occurrences and their variants in a large set of experimentally detected protein sequences based on state-of-the-art sequence matching and alignment algorithms. After detection, the method generates all the potential cleaved fragments by a specific protease. This space and time-efficient algorithm is flexible to handle the different orientations that the consensus sequence and the protein sequence can take before cleaving. It is O(mn) in space complexity and O(Nmn) in time complexity, with N number of protein sequences, m length of the consensus sequence, and n length of each protein sequence. Ultimately, this knowledge will subsequently feed into the development of a novel tool for researchers to detect diverse types of selected BDPs as putative disease markers, contributing to the diagnosis and treatment of related disorders.

  4. 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.

  5. PASTA: Ultra-Large Multiple Sequence Alignment for Nucleotide and Amino-Acid Sequences

    PubMed Central

    Mirarab, Siavash; Nguyen, Nam; Guo, Sheng; Wang, Li-San; Kim, Junhyong

    2015-01-01

    Abstract We introduce PASTA, a new multiple sequence alignment algorithm. PASTA uses a new technique to produce an alignment given a guide tree that enables it to be both highly scalable and very accurate. We present a study on biological and simulated data with up to 200,000 sequences, showing that PASTA produces highly accurate alignments, improving on the accuracy and scalability of the leading alignment methods (including SATé). We also show that trees estimated on PASTA alignments are highly accurate—slightly better than SATé trees, but with substantial improvements relative to other methods. Finally, PASTA is faster than SATé, highly parallelizable, and requires relatively little memory. PMID:25549288

  6. CaMV-35S promoter sequence-specific DNA methylation in lettuce.

    PubMed

    Okumura, Azusa; Shimada, Asahi; Yamasaki, Satoshi; Horino, Takuya; Iwata, Yuji; Koizumi, Nozomu; Nishihara, Masahiro; Mishiba, Kei-ichiro

    2016-01-01

    We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

  7. Ultraaccurate genome sequencing and haplotyping of single human cells.

    PubMed

    Chu, Wai Keung; Edge, Peter; Lee, Ho Suk; Bansal, Vikas; Bafna, Vineet; Huang, Xiaohua; Zhang, Kun

    2017-11-21

    Accurate detection of variants and long-range haplotypes in genomes of single human cells remains very challenging. Common approaches require extensive in vitro amplification of genomes of individual cells using DNA polymerases and high-throughput short-read DNA sequencing. These approaches have two notable drawbacks. First, polymerase replication errors could generate tens of thousands of false-positive calls per genome. Second, relatively short sequence reads contain little to no haplotype information. Here we report a method, which is dubbed SISSOR (single-stranded sequencing using microfluidic reactors), for accurate single-cell genome sequencing and haplotyping. A microfluidic processor is used to separate the Watson and Crick strands of the double-stranded chromosomal DNA in a single cell and to randomly partition megabase-size DNA strands into multiple nanoliter compartments for amplification and construction of barcoded libraries for sequencing. The separation and partitioning of large single-stranded DNA fragments of the homologous chromosome pairs allows for the independent sequencing of each of the complementary and homologous strands. This enables the assembly of long haplotypes and reduction of sequence errors by using the redundant sequence information and haplotype-based error removal. We demonstrated the ability to sequence single-cell genomes with error rates as low as 10 -8 and average 500-kb-long DNA fragments that can be assembled into haplotype contigs with N50 greater than 7 Mb. The performance could be further improved with more uniform amplification and more accurate sequence alignment. The ability to obtain accurate genome sequences and haplotype information from single cells will enable applications of genome sequencing for diverse clinical needs. Copyright © 2017 the Author(s). Published by PNAS.

  8. DNA sequence requirements for the accurate transcription of a protein-coding plastid gene in a plastid in vitro system from mustard (Sinapis alba L.)

    PubMed Central

    Link, Gerhard

    1984-01-01

    A nuclease-treated plastid extract from mustard (Sinapis alba L.) allows efficient transcription of cloned plastid DNA templates. In this in vitro system, the major runoff transcript of the truncated gene for the 32 000 mol. wt. photosystem II protein was accurately initiated from a site close to or identical with the in vivo start site. By using plasmids with deletions in the 5'-flanking region of this gene as templates, a DNA region required for efficient and selective initiation was detected ˜28-35 nucleotides upstream of the transcription start site. This region contains the sequence element TTGACA, which matches the consensus sequence for prokaryotic `−35' promoter elements. In the absence of this region, a region ˜13-27 nucleotides upstream of the start site still enables a basic level of specific transcription. This second region contains the sequence element TATATAA, which matches the consensus sequence for the `TATA' box of genes transcribed by RNA polymerase II (or B). The region between the `TATA'-like element and the transcription start site is not sufficient but may be required for specific transcription of the plastid gene. This latter region contains the sequence element TATACT, which resembles the prokaryotic `−10' (Pribnow) box. Based on the structural and transcriptional features of the 5' upstream region, a `promoter switch' mechanism is proposed, which may account for the developmentally regulated expression of this plastid gene. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Figure 5. PMID:16453540

  9. Learning a Weighted Sequence Model of the Nucleosome Core and Linker Yields More Accurate Predictions in Saccharomyces cerevisiae and Homo sapiens

    PubMed Central

    Reynolds, Sheila M.; Bilmes, Jeff A.; Noble, William Stafford

    2010-01-01

    DNA in eukaryotes is packaged into a chromatin complex, the most basic element of which is the nucleosome. The precise positioning of the nucleosome cores allows for selective access to the DNA, and the mechanisms that control this positioning are important pieces of the gene expression puzzle. We describe a large-scale nucleosome pattern that jointly characterizes the nucleosome core and the adjacent linkers and is predominantly characterized by long-range oscillations in the mono, di- and tri-nucleotide content of the DNA sequence, and we show that this pattern can be used to predict nucleosome positions in both Homo sapiens and Saccharomyces cerevisiae more accurately than previously published methods. Surprisingly, in both H. sapiens and S. cerevisiae, the most informative individual features are the mono-nucleotide patterns, although the inclusion of di- and tri-nucleotide features results in improved performance. Our approach combines a much longer pattern than has been previously used to predict nucleosome positioning from sequence—301 base pairs, centered at the position to be scored—with a novel discriminative classification approach that selectively weights the contributions from each of the input features. The resulting scores are relatively insensitive to local AT-content and can be used to accurately discriminate putative dyad positions from adjacent linker regions without requiring an additional dynamic programming step and without the attendant edge effects and assumptions about linker length modeling and overall nucleosome density. Our approach produces the best dyad-linker classification results published to date in H. sapiens, and outperforms two recently published models on a large set of S. cerevisiae nucleosome positions. Our results suggest that in both genomes, a comparable and relatively small fraction of nucleosomes are well-positioned and that these positions are predictable based on sequence alone. We believe that the bulk of the

  10. Accurate predictions of population-level changes in sequence and structural properties of HIV-1 Env using a volatility-controlled diffusion model

    PubMed Central

    DeLeon, Orlando; Hodis, Hagit; O’Malley, Yunxia; Johnson, Jacklyn; Salimi, Hamid; Zhai, Yinjie; Winter, Elizabeth; Remec, Claire; Eichelberger, Noah; Van Cleave, Brandon; Puliadi, Ramya; Harrington, Robert D.; Stapleton, Jack T.; Haim, Hillel

    2017-01-01

    The envelope glycoproteins (Envs) of HIV-1 continuously evolve in the host by random mutations and recombination events. The resulting diversity of Env variants circulating in the population and their continuing diversification process limit the efficacy of AIDS vaccines. We examined the historic changes in Env sequence and structural features (measured by integrity of epitopes on the Env trimer) in a geographically defined population in the United States. As expected, many Env features were relatively conserved during the 1980s. From this state, some features diversified whereas others remained conserved across the years. We sought to identify “clues” to predict the observed historic diversification patterns. Comparison of viruses that cocirculate in patients at any given time revealed that each feature of Env (sequence or structural) exists at a defined level of variance. The in-host variance of each feature is highly conserved among individuals but can vary between different HIV-1 clades. We designate this property “volatility” and apply it to model evolution of features as a linear diffusion process that progresses with increasing genetic distance. Volatilities of different features are highly correlated with their divergence in longitudinally monitored patients. Volatilities of features also correlate highly with their population-level diversification. Using volatility indices measured from a small number of patient samples, we accurately predict the population diversity that developed for each feature over the course of 30 years. Amino acid variants that evolved at key antigenic sites are also predicted well. Therefore, small “fluctuations” in feature values measured in isolated patient samples accurately describe their potential for population-level diversification. These tools will likely contribute to the design of population-targeted AIDS vaccines by effectively capturing the diversity of currently circulating strains and addressing properties

  11. MAFsnp: A Multi-Sample Accurate and Flexible SNP Caller Using Next-Generation Sequencing Data

    PubMed Central

    Hu, Jiyuan; Li, Tengfei; Xiu, Zidi; Zhang, Hong

    2015-01-01

    Most existing statistical methods developed for calling single nucleotide polymorphisms (SNPs) using next-generation sequencing (NGS) data are based on Bayesian frameworks, and there does not exist any SNP caller that produces p-values for calling SNPs in a frequentist framework. To fill in this gap, we develop a new method MAFsnp, a Multiple-sample based Accurate and Flexible algorithm for calling SNPs with NGS data. MAFsnp is based on an estimated likelihood ratio test (eLRT) statistic. In practical situation, the involved parameter is very close to the boundary of the parametric space, so the standard large sample property is not suitable to evaluate the finite-sample distribution of the eLRT statistic. Observing that the distribution of the test statistic is a mixture of zero and a continuous part, we propose to model the test statistic with a novel two-parameter mixture distribution. Once the parameters in the mixture distribution are estimated, p-values can be easily calculated for detecting SNPs, and the multiple-testing corrected p-values can be used to control false discovery rate (FDR) at any pre-specified level. With simulated data, MAFsnp is shown to have much better control of FDR than the existing SNP callers. Through the application to two real datasets, MAFsnp is also shown to outperform the existing SNP callers in terms of calling accuracy. An R package “MAFsnp” implementing the new SNP caller is freely available at http://homepage.fudan.edu.cn/zhangh/softwares/. PMID:26309201

  12. Indexed variation graphs for efficient and accurate resistome profiling.

    PubMed

    Rowe, Will P M; Winn, Martyn D

    2018-05-14

    Antimicrobial resistance remains a major threat to global health. Profiling the collective antimicrobial resistance genes within a metagenome (the "resistome") facilitates greater understanding of antimicrobial resistance gene diversity and dynamics. In turn, this can allow for gene surveillance, individualised treatment of bacterial infections and more sustainable use of antimicrobials. However, resistome profiling can be complicated by high similarity between reference genes, as well as the sheer volume of sequencing data and the complexity of analysis workflows. We have developed an efficient and accurate method for resistome profiling that addresses these complications and improves upon currently available tools. Our method combines a variation graph representation of gene sets with an LSH Forest indexing scheme to allow for fast classification of metagenomic sequence reads using similarity-search queries. Subsequent hierarchical local alignment of classified reads against graph traversals enables accurate reconstruction of full-length gene sequences using a scoring scheme. We provide our implementation, GROOT, and show it to be both faster and more accurate than a current reference-dependent tool for resistome profiling. GROOT runs on a laptop and can process a typical 2 gigabyte metagenome in 2 minutes using a single CPU. Our method is not restricted to resistome profiling and has the potential to improve current metagenomic workflows. GROOT is written in Go and is available at https://github.com/will-rowe/groot (MIT license). will.rowe@stfc.ac.uk. Supplementary data are available at Bioinformatics online.

  13. MethylMeter®: bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples

    PubMed Central

    McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

    2016-01-01

    Aim: Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Materials & methods: Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter®. Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. Results: MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. Conclusion: MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas. PMID:27337298

  14. GobyWeb: Simplified Management and Analysis of Gene Expression and DNA Methylation Sequencing Data

    PubMed Central

    Dorff, Kevin C.; Chambwe, Nyasha; Zeno, Zachary; Simi, Manuele; Shaknovich, Rita; Campagne, Fabien

    2013-01-01

    We present GobyWeb, a web-based system that facilitates the management and analysis of high-throughput sequencing (HTS) projects. The software provides integrated support for a broad set of HTS analyses and offers a simple plugin extension mechanism. Analyses currently supported include quantification of gene expression for messenger and small RNA sequencing, estimation of DNA methylation (i.e., reduced bisulfite sequencing and whole genome methyl-seq), or the detection of pathogens in sequenced data. In contrast to previous analysis pipelines developed for analysis of HTS data, GobyWeb requires significantly less storage space, runs analyses efficiently on a parallel grid, scales gracefully to process tens or hundreds of multi-gigabyte samples, yet can be used effectively by researchers who are comfortable using a web browser. We conducted performance evaluations of the software and found it to either outperform or have similar performance to analysis programs developed for specialized analyses of HTS data. We found that most biologists who took a one-hour GobyWeb training session were readily able to analyze RNA-Seq data with state of the art analysis tools. GobyWeb can be obtained at http://gobyweb.campagnelab.org and is freely available for non-commercial use. GobyWeb plugins are distributed in source code and licensed under the open source LGPL3 license to facilitate code inspection, reuse and independent extensions http://github.com/CampagneLaboratory/gobyweb2-plugins. PMID:23936070

  15. Genome-wide methylation sequencing of paired primary and metastatic cell lines identifies common DNA methylation changes and a role for EBF3 as a candidate epigenetic driver of melanoma metastasis

    PubMed Central

    Chatterjee, Aniruddha; Stockwell, Peter A; Ahn, Antonio; Rodger, Euan J; Leichter, Anna L; Eccles, Michael R

    2017-01-01

    Epigenetic alterations are increasingly implicated in metastasis, whereas very few genetic mutations have been identified as authentic drivers of cancer metastasis. Yet, to date, few studies have identified metastasis-related epigenetic drivers, in part because a framework for identifying driver epigenetic changes in metastasis has not been established. Using reduced representation bisulfite sequencing (RRBS), we mapped genome-wide DNA methylation patterns in three cutaneous primary and metastatic melanoma cell line pairs to identify metastasis-related epigenetic drivers. Globally, metastatic melanoma cell lines were hypomethylated compared to the matched primary melanoma cell lines. Using whole genome RRBS we identified 75 shared (10 hyper- and 65 hypomethylated) differentially methylated fragments (DMFs), which were associated with 68 genes showing significant methylation differences. One gene, Early B Cell Factor 3 (EBF3), exhibited promoter hypermethylation in metastatic cell lines, and was validated with bisulfite sequencing and in two publicly available independent melanoma cohorts (n = 40 and 458 melanomas, respectively). We found that hypermethylation of the EBF3 promoter was associated with increased EBF3 mRNA levels in metastatic melanomas and subsequent inhibition of DNA methylation reduced EBF3 expression. RNAi-mediated knockdown of EBF3 mRNA levels decreased proliferation, migration and invasion in primary and metastatic melanoma cell lines. Overall, we have identified numerous epigenetic changes characterising metastatic melanoma cell lines, including EBF3-induced aggressive phenotypic behaviour with elevated EBF3 expression in metastatic melanoma, suggesting that EBF3 promoter hypermethylation may be a candidate epigenetic driver of metastasis. PMID:28030832

  16. Pairagon: a highly accurate, HMM-based cDNA-to-genome aligner.

    PubMed

    Lu, David V; Brown, Randall H; Arumugam, Manimozhiyan; Brent, Michael R

    2009-07-01

    The most accurate way to determine the intron-exon structures in a genome is to align spliced cDNA sequences to the genome. Thus, cDNA-to-genome alignment programs are a key component of most annotation pipelines. The scoring system used to choose the best alignment is a primary determinant of alignment accuracy, while heuristics that prevent consideration of certain alignments are a primary determinant of runtime and memory usage. Both accuracy and speed are important considerations in choosing an alignment algorithm, but scoring systems have received much less attention than heuristics. We present Pairagon, a pair hidden Markov model based cDNA-to-genome alignment program, as the most accurate aligner for sequences with high- and low-identity levels. We conducted a series of experiments testing alignment accuracy with varying sequence identity. We first created 'perfect' simulated cDNA sequences by splicing the sequences of exons in the reference genome sequences of fly and human. The complete reference genome sequences were then mutated to various degrees using a realistic mutation simulator and the perfect cDNAs were aligned to them using Pairagon and 12 other aligners. To validate these results with natural sequences, we performed cross-species alignment using orthologous transcripts from human, mouse and rat. We found that aligner accuracy is heavily dependent on sequence identity. For sequences with 100% identity, Pairagon achieved accuracy levels of >99.6%, with one quarter of the errors of any other aligner. Furthermore, for human/mouse alignments, which are only 85% identical, Pairagon achieved 87% accuracy, higher than any other aligner. Pairagon source and executables are freely available at http://mblab.wustl.edu/software/pairagon/

  17. Predicting DNA hybridization kinetics from sequence

    NASA Astrophysics Data System (ADS)

    Zhang, Jinny X.; Fang, John Z.; Duan, Wei; Wu, Lucia R.; Zhang, Angela W.; Dalchau, Neil; Yordanov, Boyan; Petersen, Rasmus; Phillips, Andrew; Zhang, David Yu

    2018-01-01

    Hybridization is a key molecular process in biology and biotechnology, but so far there is no predictive model for accurately determining hybridization rate constants based on sequence information. Here, we report a weighted neighbour voting (WNV) prediction algorithm, in which the hybridization rate constant of an unknown sequence is predicted based on similarity reactions with known rate constants. To construct this algorithm we first performed 210 fluorescence kinetics experiments to observe the hybridization kinetics of 100 different DNA target and probe pairs (36 nt sub-sequences of the CYCS and VEGF genes) at temperatures ranging from 28 to 55 °C. Automated feature selection and weighting optimization resulted in a final six-feature WNV model, which can predict hybridization rate constants of new sequences to within a factor of 3 with ∼91% accuracy, based on leave-one-out cross-validation. Accurate prediction of hybridization kinetics allows the design of efficient probe sequences for genomics research.

  18. Using structure to explore the sequence alignment space of remote homologs.

    PubMed

    Kuziemko, Andrew; Honig, Barry; Petrey, Donald

    2011-10-01

    Protein structure modeling by homology requires an accurate sequence alignment between the query protein and its structural template. However, sequence alignment methods based on dynamic programming (DP) are typically unable to generate accurate alignments for remote sequence homologs, thus limiting the applicability of modeling methods. A central problem is that the alignment that is "optimal" in terms of the DP score does not necessarily correspond to the alignment that produces the most accurate structural model. That is, the correct alignment based on structural superposition will generally have a lower score than the optimal alignment obtained from sequence. Variations of the DP algorithm have been developed that generate alternative alignments that are "suboptimal" in terms of the DP score, but these still encounter difficulties in detecting the correct structural alignment. We present here a new alternative sequence alignment method that relies heavily on the structure of the template. By initially aligning the query sequence to individual fragments in secondary structure elements and combining high-scoring fragments that pass basic tests for "modelability", we can generate accurate alignments within a small ensemble. Our results suggest that the set of sequences that can currently be modeled by homology can be greatly extended.

  19. Environmental assessment of mild bisulfite pretreatment of forest residues into fermentable sugars for biofuel production.

    PubMed

    Nwaneshiudu, Ikechukwu C; Ganguly, Indroneil; Pierobon, Francesca; Bowers, Tait; Eastin, Ivan

    2016-01-01

    Sugar production via pretreatment and enzymatic hydrolysis of cellulosic feedstock, in this case softwood harvest residues, is a critical step in the biochemical conversion pathway towards drop-in biofuels. Mild bisulfite (MBS) pretreatment is an emerging option for the breakdown and subsequent processing of biomass towards fermentable sugars. An environmental assessment of this process is critical to discern its future sustainability in the ever-changing biofuels landscape. The subsequent cradle-to-gate assessment of a proposed sugar production facility analyzes sugar made from woody biomass using MBS pretreatment across all seven impact categories (functional unit 1 kg dry mass sugar), with a specific focus on potential global warming and eutrophication impacts. The study found that the eutrophication impact (0.000201 kg N equivalent) is less than the impacts from conventional beet and cane sugars, while the global warming impact (0.353 kg CO2 equivalent) falls within the range of conventional processes. This work discusses some of the environmental impacts of designing and operating a sugar production facility that uses MBS as a method of treating cellulosic forest residuals. The impacts of each unit process in the proposed facility are highlighted. A comparison to other sugar-making process is detailed and will inform the growing biofuels literature.

  20. An accurate algorithm for the detection of DNA fragments from dilution pool sequencing experiments.

    PubMed

    Bansal, Vikas

    2018-01-01

    The short read lengths of current high-throughput sequencing technologies limit the ability to recover long-range haplotype information. Dilution pool methods for preparing DNA sequencing libraries from high molecular weight DNA fragments enable the recovery of long DNA fragments from short sequence reads. These approaches require computational methods for identifying the DNA fragments using aligned sequence reads and assembling the fragments into long haplotypes. Although a number of computational methods have been developed for haplotype assembly, the problem of identifying DNA fragments from dilution pool sequence data has not received much attention. We formulate the problem of detecting DNA fragments from dilution pool sequencing experiments as a genome segmentation problem and develop an algorithm that uses dynamic programming to optimize a likelihood function derived from a generative model for the sequence reads. This algorithm uses an iterative approach to automatically infer the mean background read depth and the number of fragments in each pool. Using simulated data, we demonstrate that our method, FragmentCut, has 25-30% greater sensitivity compared with an HMM based method for fragment detection and can also detect overlapping fragments. On a whole-genome human fosmid pool dataset, the haplotypes assembled using the fragments identified by FragmentCut had greater N50 length, 16.2% lower switch error rate and 35.8% lower mismatch error rate compared with two existing methods. We further demonstrate the greater accuracy of our method using two additional dilution pool datasets. FragmentCut is available from https://bansal-lab.github.io/software/FragmentCut. vibansal@ucsd.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  1. Deep Sequencing to Identify the Causes of Viral Encephalitis

    PubMed Central

    Chan, Benjamin K.; Wilson, Theodore; Fischer, Kael F.; Kriesel, John D.

    2014-01-01

    Deep sequencing allows for a rapid, accurate characterization of microbial DNA and RNA sequences in many types of samples. Deep sequencing (also called next generation sequencing or NGS) is being developed to assist with the diagnosis of a wide variety of infectious diseases. In this study, seven frozen brain samples from deceased subjects with recent encephalitis were investigated. RNA from each sample was extracted, randomly reverse transcribed and sequenced. The sequence analysis was performed in a blinded fashion and confirmed with pathogen-specific PCR. This analysis successfully identified measles virus sequences in two brain samples and herpes simplex virus type-1 sequences in three brain samples. No pathogen was identified in the other two brain specimens. These results were concordant with pathogen-specific PCR and partially concordant with prior neuropathological examinations, demonstrating that deep sequencing can accurately identify viral infections in frozen brain tissue. PMID:24699691

  2. CoDE-seq, an augmented whole-exome sequencing, enables the accurate detection of CNVs and mutations in Mendelian obesity and intellectual disability.

    PubMed

    Montagne, Louise; Derhourhi, Mehdi; Piton, Amélie; Toussaint, Bénédicte; Durand, Emmanuelle; Vaillant, Emmanuel; Thuillier, Dorothée; Gaget, Stefan; De Graeve, Franck; Rabearivelo, Iandry; Lansiaux, Amélie; Lenne, Bruno; Sukno, Sylvie; Desailloud, Rachel; Cnop, Miriam; Nicolescu, Ramona; Cohen, Lior; Zagury, Jean-François; Amouyal, Mélanie; Weill, Jacques; Muller, Jean; Sand, Olivier; Delobel, Bruno; Froguel, Philippe; Bonnefond, Amélie

    2018-05-16

    The molecular diagnosis of extreme forms of obesity, in which accurate detection of both copy number variations (CNVs) and point mutations, is crucial for an optimal care of the patients and genetic counseling for their families. Whole-exome sequencing (WES) has benefited considerably this molecular diagnosis, but its poor ability to detect CNVs remains a major limitation. We aimed to develop a method (CoDE-seq) enabling the accurate detection of both CNVs and point mutations in one step. CoDE-seq is based on an augmented WES method, using probes distributed uniformly throughout the genome. CoDE-seq was validated in 40 patients for whom chromosomal DNA microarray was available. CNVs and mutations were assessed in 82 children/young adults with suspected Mendelian obesity and/or intellectual disability and in their parents when available (n total  = 145). CoDE-seq not only detected all of the 97 CNVs identified by chromosomal DNA microarrays but also found 84 additional CNVs, due to a better resolution. When compared to CoDE-seq and chromosomal DNA microarrays, WES failed to detect 37% and 14% of CNVs, respectively. In the 82 patients, a likely molecular diagnosis was achieved in >30% of the patients. Half of the genetic diagnoses were explained by CNVs while the other half by mutations. CoDE-seq has proven cost-efficient and highly effective as it avoids the sequential genetic screening approaches currently used in clinical practice for the accurate detection of CNVs and point mutations. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

  3. Historian: accurate reconstruction of ancestral sequences and evolutionary rates.

    PubMed

    Holmes, Ian H

    2017-04-15

    Reconstruction of ancestral sequence histories, and estimation of parameters like indel rates, are improved by using explicit evolutionary models and summing over uncertain alignments. The previous best tool for this purpose (according to simulation benchmarks) was ProtPal, but this tool was too slow for practical use. Historian combines an efficient reimplementation of the ProtPal algorithm with performance-improving heuristics from other alignment tools. Simulation results on fidelity of rate estimation via ancestral reconstruction, along with evaluations on the structurally informed alignment dataset BAliBase 3.0, recommend Historian over other alignment tools for evolutionary applications. Historian is available at https://github.com/evoldoers/historian under the Creative Commons Attribution 3.0 US license. ihholmes+historian@gmail.com. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  4. BASIC: A Simple and Accurate Modular DNA Assembly Method.

    PubMed

    Storch, Marko; Casini, Arturo; Mackrow, Ben; Ellis, Tom; Baldwin, Geoff S

    2017-01-01

    Biopart Assembly Standard for Idempotent Cloning (BASIC) is a simple, accurate, and robust DNA assembly method. The method is based on linker-mediated DNA assembly and provides highly accurate DNA assembly with 99 % correct assemblies for four parts and 90 % correct assemblies for seven parts [1]. The BASIC standard defines a single entry vector for all parts flanked by the same prefix and suffix sequences and its idempotent nature means that the assembled construct is returned in the same format. Once a part has been adapted into the BASIC format it can be placed at any position within a BASIC assembly without the need for reformatting. This allows laboratories to grow comprehensive and universal part libraries and to share them efficiently. The modularity within the BASIC framework is further extended by the possibility of encoding ribosomal binding sites (RBS) and peptide linker sequences directly on the linkers used for assembly. This makes BASIC a highly versatile library construction method for combinatorial part assembly including the construction of promoter, RBS, gene variant, and protein-tag libraries. In comparison with other DNA assembly standards and methods, BASIC offers a simple robust protocol; it relies on a single entry vector, provides for easy hierarchical assembly, and is highly accurate for up to seven parts per assembly round [2].

  5. Comparative Analyses of DNA Methylation and Sequence Evolution Using Nasonia Genomes

    PubMed Central

    Park, Jungsun; Peng, Zuogang; Zeng, Jia; Elango, Navin; Park, Taesung; Wheeler, Dave; Werren, John H.; Yi, Soojin V.

    2011-01-01

    The functional and evolutionary significance of DNA methylation in insect genomes remains to be resolved. Nasonia is well situated for comparative analyses of DNA methylation and genome evolution, since the genomes of a moderately distant outgroup species as well as closely related sibling species are available. Using direct sequencing of bisulfite-converted DNA, we uncovered a substantial level of DNA methylation in 17 of 18 Nasonia vitripennis genes and a strong correlation between methylation level and CpG depletion. Notably, in the sex-determining locus transformer, the exon that is alternatively spliced between the sexes is heavily methylated in both males and females, whereas other exons are only sparsely methylated. Orthologous genes of the honeybee and Nasonia show highly similar relative levels of CpG depletion, despite ∼190 My divergence. Densely and sparsely methylated genes in these species also exhibit similar functional enrichments. We found that the degree of CpG depletion is negatively correlated with substitution rates between closely related Nasonia species for synonymous, nonsynonymous, and intron sites. This suggests that mutation rates increase with decreasing levels of germ line methylation. Thus, DNA methylation is prevalent in the Nasonia genome, may participate in regulatory processes such as sex determination and alternative splicing, and is correlated with several aspects of genome and sequence evolution. PMID:21693438

  6. Neural network and SVM classifiers accurately predict lipid binding proteins, irrespective of sequence homology.

    PubMed

    Bakhtiarizadeh, Mohammad Reza; Moradi-Shahrbabak, Mohammad; Ebrahimi, Mansour; Ebrahimie, Esmaeil

    2014-09-07

    Due to the central roles of lipid binding proteins (LBPs) in many biological processes, sequence based identification of LBPs is of great interest. The major challenge is that LBPs are diverse in sequence, structure, and function which results in low accuracy of sequence homology based methods. Therefore, there is a need for developing alternative functional prediction methods irrespective of sequence similarity. To identify LBPs from non-LBPs, the performances of support vector machine (SVM) and neural network were compared in this study. Comprehensive protein features and various techniques were employed to create datasets. Five-fold cross-validation (CV) and independent evaluation (IE) tests were used to assess the validity of the two methods. The results indicated that SVM outperforms neural network. SVM achieved 89.28% (CV) and 89.55% (IE) overall accuracy in identification of LBPs from non-LBPs and 92.06% (CV) and 92.90% (IE) (in average) for classification of different LBPs classes. Increasing the number and the range of extracted protein features as well as optimization of the SVM parameters significantly increased the efficiency of LBPs class prediction in comparison to the only previous report in this field. Altogether, the results showed that the SVM algorithm can be run on broad, computationally calculated protein features and offers a promising tool in detection of LBPs classes. The proposed approach has the potential to integrate and improve the common sequence alignment based methods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Metagenome assembly through clustering of next-generation sequencing data using protein sequences.

    PubMed

    Sim, Mikang; Kim, Jaebum

    2015-02-01

    The study of environmental microbial communities, called metagenomics, has gained a lot of attention because of the recent advances in next-generation sequencing (NGS) technologies. Microbes play a critical role in changing their environments, and the mode of their effect can be solved by investigating metagenomes. However, the difficulty of metagenomes, such as the combination of multiple microbes and different species abundance, makes metagenome assembly tasks more challenging. In this paper, we developed a new metagenome assembly method by utilizing protein sequences, in addition to the NGS read sequences. Our method (i) builds read clusters by using mapping information against available protein sequences, and (ii) creates contig sequences by finding consensus sequences through probabilistic choices from the read clusters. By using simulated NGS read sequences from real microbial genome sequences, we evaluated our method in comparison with four existing assembly programs. We found that our method could generate relatively long and accurate metagenome assemblies, indicating that the idea of using protein sequences, as a guide for the assembly, is promising. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. A rapid, naked-eye detection of hypochlorite and bisulfite using a robust and highly-photostable indicator dye Quinaldine Red in aqueous medium

    NASA Astrophysics Data System (ADS)

    Dutta, Tanoy; Chandra, Falguni; Koner, Apurba L.

    2018-02-01

    A ;naked-eye; detection of health hazardous bisulfite (HSO3-) and hypochlorite (ClO-) using an indicator dye (Quinaldine Red, QR) in a wide range of pH is demonstrated. The molecule contains a quinoline moiety linked to an N,N-dimethylaniline moiety with a conjugated double bond. Treatment of QR with HSO3- and ClO-, in aqueous solution at near-neutral pH, resulted in a colorless product with high selectivity and sensitivity. The detection limit was 47.8 μM and 0.2 μM for HSO3- and ClO- respectively. However, ClO- was 50 times more sensitive and with 2 times faster response compared to HSO3-. The detail characterization and related analysis demonstrate the potential of QR for a rapid, robust and highly efficient colorimetric sensor for the practical applications to detect hypochlorite in water samples.

  9. BAC sequencing using pooled methods.

    PubMed

    Saski, Christopher A; Feltus, F Alex; Parida, Laxmi; Haiminen, Niina

    2015-01-01

    Shotgun sequencing and assembly of a large, complex genome can be both expensive and challenging to accurately reconstruct the true genome sequence. Repetitive DNA arrays, paralogous sequences, polyploidy, and heterozygosity are main factors that plague de novo genome sequencing projects that typically result in highly fragmented assemblies and are difficult to extract biological meaning. Targeted, sub-genomic sequencing offers complexity reduction by removing distal segments of the genome and a systematic mechanism for exploring prioritized genomic content through BAC sequencing. If one isolates and sequences the genome fraction that encodes the relevant biological information, then it is possible to reduce overall sequencing costs and efforts that target a genomic segment. This chapter describes the sub-genome assembly protocol for an organism based upon a BAC tiling path derived from a genome-scale physical map or from fine mapping using BACs to target sub-genomic regions. Methods that are described include BAC isolation and mapping, DNA sequencing, and sequence assembly.

  10. Multilocus sequence typing of total-genome-sequenced bacteria.

    PubMed

    Larsen, Mette V; Cosentino, Salvatore; Rasmussen, Simon; Friis, Carsten; Hasman, Henrik; Marvig, Rasmus Lykke; Jelsbak, Lars; Sicheritz-Pontén, Thomas; Ussery, David W; Aarestrup, Frank M; Lund, Ole

    2012-04-01

    Accurate strain identification is essential for anyone working with bacteria. For many species, multilocus sequence typing (MLST) is considered the "gold standard" of typing, but it is traditionally performed in an expensive and time-consuming manner. As the costs of whole-genome sequencing (WGS) continue to decline, it becomes increasingly available to scientists and routine diagnostic laboratories. Currently, the cost is below that of traditional MLST. The new challenges will be how to extract the relevant information from the large amount of data so as to allow for comparison over time and between laboratories. Ideally, this information should also allow for comparison to historical data. We developed a Web-based method for MLST of 66 bacterial species based on WGS data. As input, the method uses short sequence reads from four sequencing platforms or preassembled genomes. Updates from the MLST databases are downloaded monthly, and the best-matching MLST alleles of the specified MLST scheme are found using a BLAST-based ranking method. The sequence type is then determined by the combination of alleles identified. The method was tested on preassembled genomes from 336 isolates covering 56 MLST schemes, on short sequence reads from 387 isolates covering 10 schemes, and on a small test set of short sequence reads from 29 isolates for which the sequence type had been determined by traditional methods. The method presented here enables investigators to determine the sequence types of their isolates on the basis of WGS data. This method is publicly available at www.cbs.dtu.dk/services/MLST.

  11. Device accurately measures and records low gas-flow rates

    NASA Technical Reports Server (NTRS)

    Branum, L. W.

    1966-01-01

    Free-floating piston in a vertical column accurately measures and records low gas-flow rates. The system may be calibrated, using an adjustable flow-rate gas supply, a low pressure gage, and a sequence recorder. From the calibration rates, a nomograph may be made for easy reduction. Temperature correction may be added for further accuracy.

  12. MSeq-CNV: accurate detection of Copy Number Variation from Sequencing of Multiple samples.

    PubMed

    Malekpour, Seyed Amir; Pezeshk, Hamid; Sadeghi, Mehdi

    2018-03-05

    Currently a few tools are capable of detecting genome-wide Copy Number Variations (CNVs) based on sequencing of multiple samples. Although aberrations in mate pair insertion sizes provide additional hints for the CNV detection based on multiple samples, the majority of the current tools rely only on the depth of coverage. Here, we propose a new algorithm (MSeq-CNV) which allows detecting common CNVs across multiple samples. MSeq-CNV applies a mixture density for modeling aberrations in depth of coverage and abnormalities in the mate pair insertion sizes. Each component in this mixture density applies a Binomial distribution for modeling the number of mate pairs with aberration in the insertion size and also a Poisson distribution for emitting the read counts, in each genomic position. MSeq-CNV is applied on simulated data and also on real data of six HapMap individuals with high-coverage sequencing, in 1000 Genomes Project. These individuals include a CEU trio of European ancestry and a YRI trio of Nigerian ethnicity. Ancestry of these individuals is studied by clustering the identified CNVs. MSeq-CNV is also applied for detecting CNVs in two samples with low-coverage sequencing in 1000 Genomes Project and six samples form the Simons Genome Diversity Project.

  13. Methylation Integration (Mint) | Informatics Technology for Cancer Research (ITCR)

    Cancer.gov

    A comprehensive software pipeline and set of Galaxy tools/workflows for integrative analysis of genome-wide DNA methylation and hydroxymethylation data. Data types can be either bisulfite sequencing and/or pull-down methods.

  14. Fast and accurate phylogeny reconstruction using filtered spaced-word matches

    PubMed Central

    Sohrabi-Jahromi, Salma; Morgenstern, Burkhard

    2017-01-01

    Abstract Motivation: Word-based or ‘alignment-free’ algorithms are increasingly used for phylogeny reconstruction and genome comparison, since they are much faster than traditional approaches that are based on full sequence alignments. Existing alignment-free programs, however, are less accurate than alignment-based methods. Results: We propose Filtered Spaced Word Matches (FSWM), a fast alignment-free approach to estimate phylogenetic distances between large genomic sequences. For a pre-defined binary pattern of match and don’t-care positions, FSWM rapidly identifies spaced word-matches between input sequences, i.e. gap-free local alignments with matching nucleotides at the match positions and with mismatches allowed at the don’t-care positions. We then estimate the number of nucleotide substitutions per site by considering the nucleotides aligned at the don’t-care positions of the identified spaced-word matches. To reduce the noise from spurious random matches, we use a filtering procedure where we discard all spaced-word matches for which the overall similarity between the aligned segments is below a threshold. We show that our approach can accurately estimate substitution frequencies even for distantly related sequences that cannot be analyzed with existing alignment-free methods; phylogenetic trees constructed with FSWM distances are of high quality. A program run on a pair of eukaryotic genomes of a few hundred Mb each takes a few minutes. Availability and Implementation: The program source code for FSWM including a documentation, as well as the software that we used to generate artificial genome sequences are freely available at http://fswm.gobics.de/ Contact: chris.leimeister@stud.uni-goettingen.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:28073754

  15. Fast and accurate phylogeny reconstruction using filtered spaced-word matches.

    PubMed

    Leimeister, Chris-André; Sohrabi-Jahromi, Salma; Morgenstern, Burkhard

    2017-04-01

    Word-based or 'alignment-free' algorithms are increasingly used for phylogeny reconstruction and genome comparison, since they are much faster than traditional approaches that are based on full sequence alignments. Existing alignment-free programs, however, are less accurate than alignment-based methods. We propose Filtered Spaced Word Matches (FSWM) , a fast alignment-free approach to estimate phylogenetic distances between large genomic sequences. For a pre-defined binary pattern of match and don't-care positions, FSWM rapidly identifies spaced word-matches between input sequences, i.e. gap-free local alignments with matching nucleotides at the match positions and with mismatches allowed at the don't-care positions. We then estimate the number of nucleotide substitutions per site by considering the nucleotides aligned at the don't-care positions of the identified spaced-word matches. To reduce the noise from spurious random matches, we use a filtering procedure where we discard all spaced-word matches for which the overall similarity between the aligned segments is below a threshold. We show that our approach can accurately estimate substitution frequencies even for distantly related sequences that cannot be analyzed with existing alignment-free methods; phylogenetic trees constructed with FSWM distances are of high quality. A program run on a pair of eukaryotic genomes of a few hundred Mb each takes a few minutes. The program source code for FSWM including a documentation, as well as the software that we used to generate artificial genome sequences are freely available at http://fswm.gobics.de/. chris.leimeister@stud.uni-goettingen.de. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

  16. Next-generation sequencing identifies major DNA methylation changes during progression of Ph+ chronic myeloid leukemia

    PubMed Central

    Heller, G; Topakian, T; Altenberger, C; Cerny-Reiterer, S; Herndlhofer, S; Ziegler, B; Datlinger, P; Byrgazov, K; Bock, C; Mannhalter, C; Hörmann, G; Sperr, W R; Lion, T; Zielinski, C C; Valent, P; Zöchbauer-Müller, S

    2016-01-01

    Little is known about the impact of DNA methylation on the evolution/progression of Ph+ chronic myeloid leukemia (CML). We investigated the methylome of CML patients in chronic phase (CP-CML), accelerated phase (AP-CML) and blast crisis (BC-CML) as well as in controls by reduced representation bisulfite sequencing. Although only ~600 differentially methylated CpG sites were identified in samples obtained from CP-CML patients compared with controls, ~6500 differentially methylated CpG sites were found in samples from BC-CML patients. In the majority of affected CpG sites, methylation was increased. In CP-CML patients who progressed to AP-CML/BC-CML, we identified up to 897 genes that were methylated at the time of progression but not at the time of diagnosis. Using RNA-sequencing, we observed downregulated expression of many of these genes in BC-CML compared with CP-CML samples. Several of them are well-known tumor-suppressor genes or regulators of cell proliferation, and gene re-expression was observed by the use of epigenetic active drugs. Together, our results demonstrate that CpG site methylation clearly increases during CML progression and that it may provide a useful basis for revealing new targets of therapy in advanced CML. PMID:27211271

  17. Accurate identification of RNA editing sites from primitive sequence with deep neural networks.

    PubMed

    Ouyang, Zhangyi; Liu, Feng; Zhao, Chenghui; Ren, Chao; An, Gaole; Mei, Chuan; Bo, Xiaochen; Shu, Wenjie

    2018-04-16

    RNA editing is a post-transcriptional RNA sequence alteration. Current methods have identified editing sites and facilitated research but require sufficient genomic annotations and prior-knowledge-based filtering steps, resulting in a cumbersome, time-consuming identification process. Moreover, these methods have limited generalizability and applicability in species with insufficient genomic annotations or in conditions of limited prior knowledge. We developed DeepRed, a deep learning-based method that identifies RNA editing from primitive RNA sequences without prior-knowledge-based filtering steps or genomic annotations. DeepRed achieved 98.1% and 97.9% area under the curve (AUC) in training and test sets, respectively. We further validated DeepRed using experimentally verified U87 cell RNA-seq data, achieving 97.9% positive predictive value (PPV). We demonstrated that DeepRed offers better prediction accuracy and computational efficiency than current methods with large-scale, mass RNA-seq data. We used DeepRed to assess the impact of multiple factors on editing identification with RNA-seq data from the Association of Biomolecular Resource Facilities and Sequencing Quality Control projects. We explored developmental RNA editing pattern changes during human early embryogenesis and evolutionary patterns in Drosophila species and the primate lineage using DeepRed. Our work illustrates DeepRed's state-of-the-art performance; it may decipher the hidden principles behind RNA editing, making editing detection convenient and effective.

  18. Accurate detection for a wide range of mutation and editing sites of microRNAs from small RNA high-throughput sequencing profiles

    PubMed Central

    Zheng, Yun; Ji, Bo; Song, Renhua; Wang, Shengpeng; Li, Ting; Zhang, Xiaotuo; Chen, Kun; Li, Tianqing; Li, Jinyan

    2016-01-01

    Various types of mutation and editing (M/E) events in microRNAs (miRNAs) can change the stabilities of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles can contain many mutated and edited miRNAs. Systematic detection of miRNA mutation and editing sites from the huge volume of sRNA HTS profiles is computationally difficult, as high sensitivity and low false positive rate (FPR) are both required. We propose a novel method (named MiRME) for an accurate and fast detection of miRNA M/E sites using a progressive sequence alignment approach which refines sensitivity and improves FPR step-by-step. From 70 sRNA HTS profiles with over 1.3 billion reads, MiRME has detected thousands of statistically significant M/E sites, including 3′-editing sites, 57 A-to-I editing sites (of which 32 are novel), as well as some putative non-canonical editing sites. We demonstrated that a few non-canonical editing sites were not resulted from mutations in genome by integrating the analysis of genome HTS profiles of two human cell lines, suggesting the existence of new editing types to further diversify the functions of miRNAs. Compared with six existing studies or methods, MiRME has shown much superior performance for the identification and visualization of the M/E sites of miRNAs from the ever-increasing sRNA HTS profiles. PMID:27229138

  19. rpoB Gene Sequencing for Identification of Corynebacterium Species

    PubMed Central

    Khamis, Atieh; Raoult, Didier; La Scola, Bernard

    2004-01-01

    The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species. PMID:15364970

  20. Sequence History Update Tool

    NASA Technical Reports Server (NTRS)

    Khanampompan, Teerapat; Gladden, Roy; Fisher, Forest; DelGuercio, Chris

    2008-01-01

    The Sequence History Update Tool performs Web-based sequence statistics archiving for Mars Reconnaissance Orbiter (MRO). Using a single UNIX command, the software takes advantage of sequencing conventions to automatically extract the needed statistics from multiple files. This information is then used to populate a PHP database, which is then seamlessly formatted into a dynamic Web page. This tool replaces a previous tedious and error-prone process of manually editing HTML code to construct a Web-based table. Because the tool manages all of the statistics gathering and file delivery to and from multiple data sources spread across multiple servers, there is also a considerable time and effort savings. With the use of The Sequence History Update Tool what previously took minutes is now done in less than 30 seconds, and now provides a more accurate archival record of the sequence commanding for MRO.

  1. Panel-based Genetic Diagnostic Testing for Inherited Eye Diseases is Highly Accurate and Reproducible and More Sensitive for Variant Detection Than Exome Sequencing

    PubMed Central

    Bujakowska, Kinga M.; Sousa, Maria E.; Fonseca-Kelly, Zoë D.; Taub, Daniel G.; Janessian, Maria; Wang, Dan Yi; Au, Elizabeth D.; Sims, Katherine B.; Sweetser, David A.; Fulton, Anne B.; Liu, Qin; Wiggs, Janey L.; Gai, Xiaowu; Pierce, Eric A.

    2015-01-01

    Purpose Next-generation sequencing (NGS) based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques have not been fully defined with regard to test accuracy and reproducibility. Methods We developed a targeted enrichment and NGS approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy and glaucoma. In preparation for providing this Genetic Eye Disease (GEDi) test on a CLIA-certified basis, we performed experiments to measure the sensitivity, specificity, reproducibility as well as the clinical sensitivity of the test. Results The GEDi test is highly reproducible and accurate, with sensitivity and specificity for single nucleotide variant detection of 97.9% and 100%, respectively. The sensitivity for variant detection was notably better than the 88.3% achieved by whole exome sequencing (WES) using the same metrics, due to better coverage of targeted genes in the GEDi test compared to commercially available exome capture sets. Prospective testing of 192 patients with IRDs indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%. Conclusion The data suggest that based on quantified performance metrics, selective targeted enrichment is preferable to WES for genetic diagnostic testing. PMID:25412400

  2. Strelka: accurate somatic small-variant calling from sequenced tumor-normal sample pairs.

    PubMed

    Saunders, Christopher T; Wong, Wendy S W; Swamy, Sajani; Becq, Jennifer; Murray, Lisa J; Cheetham, R Keira

    2012-07-15

    Whole genome and exome sequencing of matched tumor-normal sample pairs is becoming routine in cancer research. The consequent increased demand for somatic variant analysis of paired samples requires methods specialized to model this problem so as to sensitively call variants at any practical level of tumor impurity. We describe Strelka, a method for somatic SNV and small indel detection from sequencing data of matched tumor-normal samples. The method uses a novel Bayesian approach which represents continuous allele frequencies for both tumor and normal samples, while leveraging the expected genotype structure of the normal. This is achieved by representing the normal sample as a mixture of germline variation with noise, and representing the tumor sample as a mixture of the normal sample with somatic variation. A natural consequence of the model structure is that sensitivity can be maintained at high tumor impurity without requiring purity estimates. We demonstrate that the method has superior accuracy and sensitivity on impure samples compared with approaches based on either diploid genotype likelihoods or general allele-frequency tests. The Strelka workflow source code is available at ftp://strelka@ftp.illumina.com/. csaunders@illumina.com

  3. 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.

  4. 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

  5. Induction and maintenance of DNA methylation in plant promoter sequences by apple latent spherical virus-induced transcriptional gene silencing

    PubMed Central

    Kon, Tatsuya; Yoshikawa, Nobuyuki

    2014-01-01

    Apple latent spherical virus (ALSV) is an efficient virus-induced gene silencing vector in functional genomics analyses of a broad range of plant species. Here, an Agrobacterium-mediated inoculation (agroinoculation) system was developed for the ALSV vector, and virus-induced transcriptional gene silencing (VITGS) is described in plants infected with the ALSV vector. The cDNAs of ALSV RNA1 and RNA2 were inserted between the cauliflower mosaic virus 35S promoter and the NOS-T sequences in a binary vector pCAMBIA1300 to produce pCALSR1 and pCALSR2-XSB or pCALSR2-XSB/MN. When these vector constructs were agroinoculated into Nicotiana benthamiana plants with a construct expressing a viral silencing suppressor, the infection efficiency of the vectors was 100%. A recombinant ALSV vector carrying part of the 35S promoter sequence induced transcriptional gene silencing of the green fluorescent protein gene in a line of N. benthamiana plants, resulting in the disappearance of green fluorescence of infected plants. Bisulfite sequencing showed that cytosine residues at CG and CHG sites of the 35S promoter sequence were highly methylated in the silenced generation zero plants infected with the ALSV carrying the promoter sequence as well as in progeny. The ALSV-mediated VITGS state was inherited by progeny for multiple generations. In addition, induction of VITGS of an endogenous gene (chalcone synthase-A) was demonstrated in petunia plants infected with an ALSV vector carrying the native promoter sequence. These results suggest that ALSV-based vectors can be applied to study DNA methylation in plant genomes, and provide a useful tool for plant breeding via epigenetic modification. PMID:25426109

  6. Sequencing technologies - the next generation.

    PubMed

    Metzker, Michael L

    2010-01-01

    Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest.

  7. Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes

    PubMed Central

    Shiroguchi, Katsuyuki; Jia, Tony Z.; Sims, Peter A.; Xie, X. Sunney

    2012-01-01

    RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq. PMID:22232676

  8. Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp.

    PubMed Central

    Tyson, G. H.; Chen, Y.; Li, C.; Mukherjee, S.; Young, S.; Lam, C.; Folster, J. P.; Whichard, J. M.; McDermott, P. F.

    2015-01-01

    The objectives of this study were to identify antimicrobial resistance genotypes for Campylobacter and to evaluate the correlation between resistance phenotypes and genotypes using in vitro antimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114 Campylobacter species isolates (82 C. coli and 32 C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, including tet(O), blaOXA-61, catA, lnu(C), aph(2″)-Ib, aph(2″)-Ic, aph(2′)-If, aph(2″)-Ig, aph(2″)-Ih, aac(6′)-Ie-aph(2″)-Ia, aac(6′)-Ie-aph(2″)-If, aac(6′)-Im, aadE, sat4, ant(6′), aad9, aph(3′)-Ic, and aph(3′)-IIIa, and mutations in two housekeeping genes (gyrA and 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs. PMID:26519386

  9. Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp.

    PubMed

    Zhao, S; Tyson, G H; Chen, Y; Li, C; Mukherjee, S; Young, S; Lam, C; Folster, J P; Whichard, J M; McDermott, P F

    2016-01-15

    The objectives of this study were to identify antimicrobial resistance genotypes for Campylobacter and to evaluate the correlation between resistance phenotypes and genotypes using in vitro antimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114 Campylobacter species isolates (82 C. coli and 32 C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, including tet(O), blaOXA-61, catA, lnu(C), aph(2″)-Ib, aph(2″)-Ic, aph(2')-If, aph(2″)-Ig, aph(2″)-Ih, aac(6')-Ie-aph(2″)-Ia, aac(6')-Ie-aph(2″)-If, aac(6')-Im, aadE, sat4, ant(6'), aad9, aph(3')-Ic, and aph(3')-IIIa, and mutations in two housekeeping genes (gyrA and 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  10. Children's discrimination of vowel sequences

    NASA Astrophysics Data System (ADS)

    Coady, Jeffry A.; Kluender, Keith R.; Evans, Julia

    2003-10-01

    Children's ability to discriminate sequences of steady-state vowels was investigated. Vowels (as in ``beet,'' ``bat,'' ``bought,'' and ``boot'') were synthesized at durations of 40, 80, 160, 320, 640, and 1280 ms. Four different vowel sequences were created by concatenating different orders of vowels for each duration, separated by 10-ms intervening silence. Thus, sequences differed in vowel order and duration (rate). Sequences were 12 s in duration, with amplitude ramped linearly over the first and last 2 s. Sequence pairs included both same (identical sequences) and different trials (sequences with vowels in different orders). Sequences with vowel of equal duration were presented on individual trials. Children aged 7;0 to 10;6 listened to pairs of sequences (with 100 ms between sequences) and responded whether sequences sounded the same or different. Results indicate that children are best able to discriminate sequences of intermediate-duration vowels, typical of conversational speaking rate. Children were less accurate with both shorter and longer vowels. Results are discussed in terms of auditory processing (shortest vowels) and memory (longest vowels). [Research supported by NIDCD DC-05263, DC-04072, and DC-005650.

  11. Facilitated sequence counting and assembly by template mutagenesis

    PubMed Central

    Levy, Dan; Wigler, Michael

    2014-01-01

    Presently, inferring the long-range structure of the DNA templates is limited by short read lengths. Accurate template counts suffer from distortions occurring during PCR amplification. We explore the utility of introducing random mutations in identical or nearly identical templates to create distinguishable patterns that are inherited during subsequent copying. We simulate the applications of this process under assumptions of error-free sequencing and perfect mapping, using cytosine deamination as a model for mutation. The simulations demonstrate that within readily achievable conditions of nucleotide conversion and sequence coverage, we can accurately count the number of otherwise identical molecules as well as connect variants separated by long spans of identical sequence. We discuss many potential applications, such as transcript profiling, isoform assembly, haplotype phasing, and de novo genome assembly. PMID:25313059

  12. GASP: Gapped Ancestral Sequence Prediction for proteins

    PubMed Central

    Edwards, Richard J; Shields, Denis C

    2004-01-01

    Background The prediction of ancestral protein sequences from multiple sequence alignments is useful for many bioinformatics analyses. Predicting ancestral sequences is not a simple procedure and relies on accurate alignments and phylogenies. Several algorithms exist based on Maximum Parsimony or Maximum Likelihood methods but many current implementations are unable to process residues with gaps, which may represent insertion/deletion (indel) events or sequence fragments. Results Here we present a new algorithm, GASP (Gapped Ancestral Sequence Prediction), for predicting ancestral sequences from phylogenetic trees and the corresponding multiple sequence alignments. Alignments may be of any size and contain gaps. GASP first assigns the positions of gaps in the phylogeny before using a likelihood-based approach centred on amino acid substitution matrices to assign ancestral amino acids. Important outgroup information is used by first working down from the tips of the tree to the root, using descendant data only to assign probabilities, and then working back up from the root to the tips using descendant and outgroup data to make predictions. GASP was tested on a number of simulated datasets based on real phylogenies. Prediction accuracy for ungapped data was similar to three alternative algorithms tested, with GASP performing better in some cases and worse in others. Adding simple insertions and deletions to the simulated data did not have a detrimental effect on GASP accuracy. Conclusions GASP (Gapped Ancestral Sequence Prediction) will predict ancestral sequences from multiple protein alignments of any size. Although not as accurate in all cases as some of the more sophisticated maximum likelihood approaches, it can process a wide range of input phylogenies and will predict ancestral sequences for gapped and ungapped residues alike. PMID:15350199

  13. A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2.

    PubMed

    Li, Jianli; Dai, Hongzheng; Feng, Yanming; Tang, Jia; Chen, Stella; Tian, Xia; Gorman, Elizabeth; Schmitt, Eric S; Hansen, Terah A A; Wang, Jing; Plon, Sharon E; Zhang, Victor Wei; Wong, Lee-Jun C

    2015-09-01

    Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3' region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  14. In silico segmentations of lentivirus envelope sequences

    PubMed Central

    Boissin-Quillon, Aurélia; Piau, Didier; Leroux, Caroline

    2007-01-01

    Background The gene encoding the envelope of lentiviruses exhibits a considerable plasticity, particularly the region which encodes the surface (SU) glycoprotein. Interestingly, mutations do not appear uniformly along the sequence of SU, but they are clustered in restricted areas, called variable (V) regions, which are interspersed with relatively more stable regions, called constant (C) regions. We look for specific signatures of C/V regions, using hidden Markov models constructed with SU sequences of the equine, human, small ruminant and simian lentiviruses. Results Our models yield clear and accurate delimitations of the C/V regions, when the test set and the training set were made up of sequences of the same lentivirus, but also when they were made up of sequences of different lentiviruses. Interestingly, the models predicted the different regions of lentiviruses such as the bovine and feline lentiviruses, not used in the training set. Models based on composite training sets produce accurate segmentations of sequences of all these lentiviruses. Conclusion Our results suggest that each C/V region has a specific statistical oligonucleotide composition, and that the C (respectively V) regions of one of these lentiviruses are statistically more similar to the C (respectively V) regions of the other lentiviruses, than to the V (respectively C) regions of the same lentivirus. PMID:17376229

  15. Accurate Simulation and Detection of Coevolution Signals in Multiple Sequence Alignments

    PubMed Central

    Ackerman, Sharon H.; Tillier, Elisabeth R.; Gatti, Domenico L.

    2012-01-01

    Background While the conserved positions of a multiple sequence alignment (MSA) are clearly of interest, non-conserved positions can also be important because, for example, destabilizing effects at one position can be compensated by stabilizing effects at another position. Different methods have been developed to recognize the evolutionary relationship between amino acid sites, and to disentangle functional/structural dependencies from historical/phylogenetic ones. Methodology/Principal Findings We have used two complementary approaches to test the efficacy of these methods. In the first approach, we have used a new program, MSAvolve, for the in silico evolution of MSAs, which records a detailed history of all covarying positions, and builds a global coevolution matrix as the accumulated sum of individual matrices for the positions forced to co-vary, the recombinant coevolution, and the stochastic coevolution. We have simulated over 1600 MSAs for 8 protein families, which reflect sequences of different sizes and proteins with widely different functions. The calculated coevolution matrices were compared with the coevolution matrices obtained for the same evolved MSAs with different coevolution detection methods. In a second approach we have evaluated the capacity of the different methods to predict close contacts in the representative X-ray structures of an additional 150 protein families using only experimental MSAs. Conclusions/Significance Methods based on the identification of global correlations between pairs were found to be generally superior to methods based only on local correlations in their capacity to identify coevolving residues using either simulated or experimental MSAs. However, the significant variability in the performance of different methods with different proteins suggests that the simulation of MSAs that replicate the statistical properties of the experimental MSA can be a valuable tool to identify the coevolution detection method that is most

  16. Optical mapping and its potential for large-scale sequencing projects.

    PubMed

    Aston, C; Mishra, B; Schwartz, D C

    1999-07-01

    Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.

  17. Entomopathogen ID: a curated sequence resource for entomopathogenic fungi

    USDA-ARS?s Scientific Manuscript database

    We report the development of a publicly accessible, curated database of Hypocrealean entomopathogenic fungi sequence data. The goal is to provide a platform for users to easily access sequence data from reference strains. The database can be used to accurately identify unknown entomopathogenic fungi...

  18. Discriminative prediction of mammalian enhancers from DNA sequence

    PubMed Central

    Lee, Dongwon; Karchin, Rachel; Beer, Michael A.

    2011-01-01

    Accurately predicting regulatory sequences and enhancers in entire genomes is an important but difficult problem, especially in large vertebrate genomes. With the advent of ChIP-seq technology, experimental detection of genome-wide EP300/CREBBP bound regions provides a powerful platform to develop predictive tools for regulatory sequences and to study their sequence properties. Here, we develop a support vector machine (SVM) framework which can accurately identify EP300-bound enhancers using only genomic sequence and an unbiased set of general sequence features. Moreover, we find that the predictive sequence features identified by the SVM classifier reveal biologically relevant sequence elements enriched in the enhancers, but we also identify other features that are significantly depleted in enhancers. The predictive sequence features are evolutionarily conserved and spatially clustered, providing further support of their functional significance. Although our SVM is trained on experimental data, we also predict novel enhancers and show that these putative enhancers are significantly enriched in both ChIP-seq signal and DNase I hypersensitivity signal in the mouse brain and are located near relevant genes. Finally, we present results of comparisons between other EP300/CREBBP data sets using our SVM and uncover sequence elements enriched and/or depleted in the different classes of enhancers. Many of these sequence features play a role in specifying tissue-specific or developmental-stage-specific enhancer activity, but our results indicate that some features operate in a general or tissue-independent manner. In addition to providing a high confidence list of enhancer targets for subsequent experimental investigation, these results contribute to our understanding of the general sequence structure of vertebrate enhancers. PMID:21875935

  19. Maternal obesity and gestational weight gain are modestly associated with umbilical cord DNA methylation

    USDA-ARS?s Scientific Manuscript database

    Maternal obesity (OB) and excessive gestational weight gain (GWG) are strong independent contributors that augment obesity risk in offspring. However, direct evidence of epigenetic changes associated with maternal habitus remains sparse. We utilized Bisulfite Amplicon Sequencing (BSAS) to conduct t...

  20. Calling Chromosome Alterations, DNA Methylation Statuses, and Mutations in Tumors by Simple Targeted Next-Generation Sequencing: A Solution for Transferring Integrated Pangenomic Studies into Routine Practice?

    PubMed

    Garinet, Simon; Néou, Mario; de La Villéon, Bruno; Faillot, Simon; Sakat, Julien; Da Fonseca, Juliana P; Jouinot, Anne; Le Tourneau, Christophe; Kamal, Maud; Luscap-Rondof, Windy; Boeva, Valentina; Gaujoux, Sebastien; Vidaud, Michel; Pasmant, Eric; Letourneur, Franck; Bertherat, Jérôme; Assié, Guillaume

    2017-09-01

    Pangenomic studies identified distinct molecular classes for many cancers, with major clinical applications. However, routine use requires cost-effective assays. We assessed whether targeted next-generation sequencing (NGS) could call chromosomal alterations and DNA methylation status. A training set of 77 tumors and a validation set of 449 (43 tumor types) were analyzed by targeted NGS and single-nucleotide polymorphism (SNP) arrays. Thirty-two tumors were analyzed by NGS after bisulfite conversion, and compared to methylation array or methylation-specific multiplex ligation-dependent probe amplification. Considering allelic ratios, correlation was strong between targeted NGS and SNP arrays (r = 0.88). In contrast, considering DNA copy number, for variations of one DNA copy, correlation was weaker between read counts and SNP array (r = 0.49). Thus, we generated TARGOMICs, optimized for detecting chromosome alterations by combining allelic ratios and read counts generated by targeted NGS. Sensitivity for calling normal, lost, and gained chromosomes was 89%, 72%, and 31%, respectively. Specificity was 81%, 93%, and 98%, respectively. These results were confirmed in the validation set. Finally, TARGOMICs could efficiently align and compute proportions of methylated cytosines from bisulfite-converted DNA from targeted NGS. In conclusion, beyond calling mutations, targeted NGS efficiently calls chromosome alterations and methylation status in tumors. A single run and minor design/protocol adaptations are sufficient. Optimizing targeted NGS should expand translation of genomics to clinical routine. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  1. RAMICS: trainable, high-speed and biologically relevant alignment of high-throughput sequencing reads to coding DNA

    PubMed Central

    Wright, Imogen A.; Travers, Simon A.

    2014-01-01

    The challenge presented by high-throughput sequencing necessitates the development of novel tools for accurate alignment of reads to reference sequences. Current approaches focus on using heuristics to map reads quickly to large genomes, rather than generating highly accurate alignments in coding regions. Such approaches are, thus, unsuited for applications such as amplicon-based analysis and the realignment phase of exome sequencing and RNA-seq, where accurate and biologically relevant alignment of coding regions is critical. To facilitate such analyses, we have developed a novel tool, RAMICS, that is tailored to mapping large numbers of sequence reads to short lengths (<10 000 bp) of coding DNA. RAMICS utilizes profile hidden Markov models to discover the open reading frame of each sequence and aligns to the reference sequence in a biologically relevant manner, distinguishing between genuine codon-sized indels and frameshift mutations. This approach facilitates the generation of highly accurate alignments, accounting for the error biases of the sequencing machine used to generate reads, particularly at homopolymer regions. Performance improvements are gained through the use of graphics processing units, which increase the speed of mapping through parallelization. RAMICS substantially outperforms all other mapping approaches tested in terms of alignment quality while maintaining highly competitive speed performance. PMID:24861618

  2. Robust temporal alignment of multimodal cardiac sequences

    NASA Astrophysics Data System (ADS)

    Perissinotto, Andrea; Queirós, Sandro; Morais, Pedro; Baptista, Maria J.; Monaghan, Mark; Rodrigues, Nuno F.; D'hooge, Jan; Vilaça, João. L.; Barbosa, Daniel

    2015-03-01

    Given the dynamic nature of cardiac function, correct temporal alignment of pre-operative models and intraoperative images is crucial for augmented reality in cardiac image-guided interventions. As such, the current study focuses on the development of an image-based strategy for temporal alignment of multimodal cardiac imaging sequences, such as cine Magnetic Resonance Imaging (MRI) or 3D Ultrasound (US). First, we derive a robust, modality-independent signal from the image sequences, estimated by computing the normalized cross-correlation between each frame in the temporal sequence and the end-diastolic frame. This signal is a resembler for the left-ventricle (LV) volume curve over time, whose variation indicates different temporal landmarks of the cardiac cycle. We then perform the temporal alignment of these surrogate signals derived from MRI and US sequences of the same patient through Dynamic Time Warping (DTW), allowing to synchronize both sequences. The proposed framework was evaluated in 98 patients, which have undergone both 3D+t MRI and US scans. The end-systolic frame could be accurately estimated as the minimum of the image-derived surrogate signal, presenting a relative error of 1.6 +/- 1.9% and 4.0 +/- 4.2% for the MRI and US sequences, respectively, thus supporting its association with key temporal instants of the cardiac cycle. The use of DTW reduces the desynchronization of the cardiac events in MRI and US sequences, allowing to temporally align multimodal cardiac imaging sequences. Overall, a generic, fast and accurate method for temporal synchronization of MRI and US sequences of the same patient was introduced. This approach could be straightforwardly used for the correct temporal alignment of pre-operative MRI information and intra-operative US images.

  3. A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum

    PubMed Central

    2014-01-01

    Background Tuber melanosporum, also known in the gastronomic community as “truffle”, features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity. Findings We performed whole-genome DNA bisulfite sequencing and mRNA sequencing on different developmental stages of T. melanosporum; namely, fruitbody (“truffle”), free-living mycelium and ectomycorrhiza. The data revealed a high rate of cytosine methylation (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. Whole genome DNA sequencing uncovered multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs, almost exclusively in free-living mycelium propagated in vitro. Treatment of mycelia with 5-azacytidine partially reduced DNA methylation and increased TE transcription. Our transcriptome assembly also resulted in the identification of a set of novel transcripts from 614 genes. Conclusions The datasets presented here provide valuable and comprehensive (epi)genomic information that can be of interest for evolutionary genomics studies of multicellular (filamentous) fungi, in particular Ascomycetes belonging to the subphylum, Pezizomycotina. Evidence derived from comparative methylome and transcriptome analyses indicates that a non-exhaustive and partly reversible methylation process operates in truffles. PMID:25392735

  4. A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum.

    PubMed

    Chen, Pao-Yang; Montanini, Barbara; Liao, Wen-Wei; Morselli, Marco; Jaroszewicz, Artur; Lopez, David; Ottonello, Simone; Pellegrini, Matteo

    2014-01-01

    Tuber melanosporum, also known in the gastronomic community as "truffle", features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity. We performed whole-genome DNA bisulfite sequencing and mRNA sequencing on different developmental stages of T. melanosporum; namely, fruitbody ("truffle"), free-living mycelium and ectomycorrhiza. The data revealed a high rate of cytosine methylation (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. Whole genome DNA sequencing uncovered multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs, almost exclusively in free-living mycelium propagated in vitro. Treatment of mycelia with 5-azacytidine partially reduced DNA methylation and increased TE transcription. Our transcriptome assembly also resulted in the identification of a set of novel transcripts from 614 genes. The datasets presented here provide valuable and comprehensive (epi)genomic information that can be of interest for evolutionary genomics studies of multicellular (filamentous) fungi, in particular Ascomycetes belonging to the subphylum, Pezizomycotina. Evidence derived from comparative methylome and transcriptome analyses indicates that a non-exhaustive and partly reversible methylation process operates in truffles.

  5. Comparative whole genome DNA methylation profiling of cattle sperm and somatic tissues reveals striking hypomethylated patterns in sperm

    USDA-ARS?s Scientific Manuscript database

    Using whole-genome bisulfite sequencing (WGBS), we profiled the DNA methylome of cattle sperms through comparison with three bovine somatic tissues (mammary grand, brain and blood). Large differences between them were observed in the methylation patterns of global CpGs, pericentromeric satellites, p...

  6. Postnatal epigenetic regulation of intestinal stem cells requires DNA methylation and is guided by the microbiome

    USDA-ARS?s Scientific Manuscript database

    DNA methylation is an epigenetic mechanism central to the development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited. We used whole genome bisulfite sequencing, and found that differentiation of mouse colonic intestinal stem cell...

  7. Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments.

    PubMed

    Povedano, Eloy; Vargas, Eva; Montiel, Víctor Ruiz-Valdepeñas; Torrente-Rodríguez, Rebeca M; Pedrero, María; Barderas, Rodrigo; Segundo-Acosta, Pablo San; Peláez-García, Alberto; Mendiola, Marta; Hardisson, David; Campuzano, Susana; Pingarrón, José M

    2018-04-23

    This paper describes two different electrochemical affinity biosensing approaches for the simple, fast and bisulfite and PCR-free quantification of 5-methylated cytosines (5-mC) in DNA using the anti-5-mC antibody as biorecognition element. One of the biosensing approaches used the anti-5-mC as capture bioreceptor and a sandwich type immunoassay, while the other one involved the use of a specific DNA probe and the anti-5-mC as a detector bioreceptor of the captured methylated DNA. Both strategies, named for simplicity in the text as immunosensor and DNA sensor, respectively, were implemented on the surface of magnetic microparticles and the transduction was accomplished by amperometry at screen-printed carbon electrodes by means of the hydrogen peroxide/hydroquinone system. The resulting amperometric biosensors demonstrated reproducibility throughout the entire protocol, sensitive determination with no need for using amplification strategies, and competitiveness with the conventional enzyme-linked immunosorbent assay methodology and the few electrochemical biosensors reported so far in terms of simplicity, sensitivity and assay time. The DNA sensor exhibited higher sensitivity and allowed the detection of the gene-specific methylations conversely to the immunosensor, which detected global DNA methylation. In addition, the DNA sensor demonstrated successful applicability for 1 h-analysis of specific methylation in two relevant tumor suppressor genes in spiked biological fluids and in genomic DNA extracted from human glioblastoma cells.

  8. Simple chained guide trees give high-quality protein multiple sequence alignments

    PubMed Central

    Boyce, Kieran; Sievers, Fabian; Higgins, Desmond G.

    2014-01-01

    Guide trees are used to decide the order of sequence alignment in the progressive multiple sequence alignment heuristic. These guide trees are often the limiting factor in making large alignments, and considerable effort has been expended over the years in making these quickly or accurately. In this article we show that, at least for protein families with large numbers of sequences that can be benchmarked with known structures, simple chained guide trees give the most accurate alignments. These also happen to be the fastest and simplest guide trees to construct, computationally. Such guide trees have a striking effect on the accuracy of alignments produced by some of the most widely used alignment packages. There is a marked increase in accuracy and a marked decrease in computational time, once the number of sequences goes much above a few hundred. This is true, even if the order of sequences in the guide tree is random. PMID:25002495

  9. RAMICS: trainable, high-speed and biologically relevant alignment of high-throughput sequencing reads to coding DNA.

    PubMed

    Wright, Imogen A; Travers, Simon A

    2014-07-01

    The challenge presented by high-throughput sequencing necessitates the development of novel tools for accurate alignment of reads to reference sequences. Current approaches focus on using heuristics to map reads quickly to large genomes, rather than generating highly accurate alignments in coding regions. Such approaches are, thus, unsuited for applications such as amplicon-based analysis and the realignment phase of exome sequencing and RNA-seq, where accurate and biologically relevant alignment of coding regions is critical. To facilitate such analyses, we have developed a novel tool, RAMICS, that is tailored to mapping large numbers of sequence reads to short lengths (<10 000 bp) of coding DNA. RAMICS utilizes profile hidden Markov models to discover the open reading frame of each sequence and aligns to the reference sequence in a biologically relevant manner, distinguishing between genuine codon-sized indels and frameshift mutations. This approach facilitates the generation of highly accurate alignments, accounting for the error biases of the sequencing machine used to generate reads, particularly at homopolymer regions. Performance improvements are gained through the use of graphics processing units, which increase the speed of mapping through parallelization. RAMICS substantially outperforms all other mapping approaches tested in terms of alignment quality while maintaining highly competitive speed performance. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. DendroBLAST: approximate phylogenetic trees in the absence of multiple sequence alignments.

    PubMed

    Kelly, Steven; Maini, Philip K

    2013-01-01

    The rapidly growing availability of genome information has created considerable demand for both fast and accurate phylogenetic inference algorithms. We present a novel method called DendroBLAST for reconstructing phylogenetic dendrograms/trees from protein sequences using BLAST. This method differs from other methods by incorporating a simple model of sequence evolution to test the effect of introducing sequence changes on the reliability of the bipartitions in the inferred tree. Using realistic simulated sequence data we demonstrate that this method produces phylogenetic trees that are more accurate than other commonly-used distance based methods though not as accurate as maximum likelihood methods from good quality multiple sequence alignments. In addition to tests on simulated data, we use DendroBLAST to generate input trees for a supertree reconstruction of the phylogeny of the Archaea. This independent analysis produces an approximate phylogeny of the Archaea that has both high precision and recall when compared to previously published analysis of the same dataset using conventional methods. Taken together these results demonstrate that approximate phylogenetic trees can be produced in the absence of multiple sequence alignments, and we propose that these trees will provide a platform for improving and informing downstream bioinformatic analysis. A web implementation of the DendroBLAST method is freely available for use at http://www.dendroblast.com/.

  11. Protein Structure Determination using Metagenome sequence data

    PubMed Central

    Ovchinnikov, Sergey; Park, Hahnbeom; Varghese, Neha; Huang, Po-Ssu; Pavlopoulos, Georgios A.; Kim, David E.; Kamisetty, Hetunandan; Kyrpides, Nikos C.; Baker, David

    2017-01-01

    Despite decades of work by structural biologists, there are still ~5200 protein families with unknown structure outside the range of comparative modeling. We show that Rosetta structure prediction guided by residue-residue contacts inferred from evolutionary information can accurately model proteins that belong to large families, and that metagenome sequence data more than triples the number of protein families with sufficient sequences for accurate modeling. We then integrate metagenome data, contact based structure matching and Rosetta structure calculations to generate models for 614 protein families with currently unknown structures; 206 are membrane proteins and 137 have folds not represented in the PDB. This approach provides the representative models for large protein families originally envisioned as the goal of the protein structure initiative at a fraction of the cost. PMID:28104891

  12. Centrifuge: rapid and sensitive classification of metagenomic sequences

    PubMed Central

    Song, Li; Breitwieser, Florian P.

    2016-01-01

    Centrifuge is a novel microbial classification engine that enables rapid, accurate, and sensitive labeling of reads and quantification of species on desktop computers. The system uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index, optimized specifically for the metagenomic classification problem. Centrifuge requires a relatively small index (4.2 GB for 4078 bacterial and 200 archaeal genomes) and classifies sequences at very high speed, allowing it to process the millions of reads from a typical high-throughput DNA sequencing run within a few minutes. Together, these advances enable timely and accurate analysis of large metagenomics data sets on conventional desktop computers. Because of its space-optimized indexing schemes, Centrifuge also makes it possible to index the entire NCBI nonredundant nucleotide sequence database (a total of 109 billion bases) with an index size of 69 GB, in contrast to k-mer-based indexing schemes, which require far more extensive space. PMID:27852649

  13. HIPPI: highly accurate protein family classification with ensembles of HMMs.

    PubMed

    Nguyen, Nam-Phuong; Nute, Michael; Mirarab, Siavash; Warnow, Tandy

    2016-11-11

    Given a new biological sequence, detecting membership in a known family is a basic step in many bioinformatics analyses, with applications to protein structure and function prediction and metagenomic taxon identification and abundance profiling, among others. Yet family identification of sequences that are distantly related to sequences in public databases or that are fragmentary remains one of the more difficult analytical problems in bioinformatics. We present a new technique for family identification called HIPPI (Hierarchical Profile Hidden Markov Models for Protein family Identification). HIPPI uses a novel technique to represent a multiple sequence alignment for a given protein family or superfamily by an ensemble of profile hidden Markov models computed using HMMER. An evaluation of HIPPI on the Pfam database shows that HIPPI has better overall precision and recall than blastp, HMMER, and pipelines based on HHsearch, and maintains good accuracy even for fragmentary query sequences and for protein families with low average pairwise sequence identity, both conditions where other methods degrade in accuracy. HIPPI provides accurate protein family identification and is robust to difficult model conditions. Our results, combined with observations from previous studies, show that ensembles of profile Hidden Markov models can better represent multiple sequence alignments than a single profile Hidden Markov model, and thus can improve downstream analyses for various bioinformatic tasks. Further research is needed to determine the best practices for building the ensemble of profile Hidden Markov models. HIPPI is available on GitHub at https://github.com/smirarab/sepp .

  14. Large-Scale Concatenation cDNA Sequencing

    PubMed Central

    Yu, Wei; Andersson, Björn; Worley, Kim C.; Muzny, Donna M.; Ding, Yan; Liu, Wen; Ricafrente, Jennifer Y.; Wentland, Meredith A.; Lennon, Greg; Gibbs, Richard A.

    1997-01-01

    A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7–2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (>20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (≥98% identity), and 16 clones generated nonexact matches (57%–97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching. [All 65 cDNA clone sequences described in this paper have been submitted to the GenBank data library under accession nos. U79240–U79304.] PMID:9110174

  15. Sequencing intractable DNA to close microbial genomes.

    PubMed

    Hurt, Richard A; Brown, Steven D; Podar, Mircea; Palumbo, Anthony V; Elias, Dwayne A

    2012-01-01

    Advancement in high throughput DNA sequencing technologies has supported a rapid proliferation of microbial genome sequencing projects, providing the genetic blueprint for in-depth studies. Oftentimes, difficult to sequence regions in microbial genomes are ruled "intractable" resulting in a growing number of genomes with sequence gaps deposited in databases. A procedure was developed to sequence such problematic regions in the "non-contiguous finished" Desulfovibrio desulfuricans ND132 genome (6 intractable gaps) and the Desulfovibrio africanus genome (1 intractable gap). The polynucleotides surrounding each gap formed GC rich secondary structures making the regions refractory to amplification and sequencing. Strand-displacing DNA polymerases used in concert with a novel ramped PCR extension cycle supported amplification and closure of all gap regions in both genomes. The developed procedures support accurate gene annotation, and provide a step-wise method that reduces the effort required for genome finishing.

  16. iMETHYL: an integrative database of human DNA methylation, gene expression, and genomic variation.

    PubMed

    Komaki, Shohei; Shiwa, Yuh; Furukawa, Ryohei; Hachiya, Tsuyoshi; Ohmomo, Hideki; Otomo, Ryo; Satoh, Mamoru; Hitomi, Jiro; Sobue, Kenji; Sasaki, Makoto; Shimizu, Atsushi

    2018-01-01

    We launched an integrative multi-omics database, iMETHYL (http://imethyl.iwate-megabank.org). iMETHYL provides whole-DNA methylation (~24 million autosomal CpG sites), whole-genome (~9 million single-nucleotide variants), and whole-transcriptome (>14 000 genes) data for CD4 + T-lymphocytes, monocytes, and neutrophils collected from approximately 100 subjects. These data were obtained from whole-genome bisulfite sequencing, whole-genome sequencing, and whole-transcriptome sequencing, making iMETHYL a comprehensive database.

  17. CpG methylation differences between neurons and glia are highly conserved from mouse to human

    USDA-ARS?s Scientific Manuscript database

    Understanding epigenetic differences that distinguish neurons and glia is of fundamental importance to the nascent field of neuroepigenetics. A recent study used genome-wide bisulfite sequencing to survey differences in DNA methylation between these two cell types, in both humans and mice. That stud...

  18. Stroma Based Prognosticators Incorporating Differences between African and European Americans

    DTIC Science & Technology

    2017-10-01

    amenable to bisulfite sequencing of more than a few genes. Exploiting the recent three-fold reduction in the cost of sequencing per read , we developed oligo...cards. The ability of the HiSeq 4000 to obtain about three times as many reads as the HiSeq2500, at the same price, means we can stay on track, though...capture, and sequencing (Table 2). We obtain tens of millions of mapped deduplicated reads per sample, while using only 5% of a sequencing lane per sample

  19. Accurate read-based metagenome characterization using a hierarchical suite of unique signatures

    PubMed Central

    Freitas, Tracey Allen K.; Li, Po-E; Scholz, Matthew B.; Chain, Patrick S. G.

    2015-01-01

    A major challenge in the field of shotgun metagenomics is the accurate identification of organisms present within a microbial community, based on classification of short sequence reads. Though existing microbial community profiling methods have attempted to rapidly classify the millions of reads output from modern sequencers, the combination of incomplete databases, similarity among otherwise divergent genomes, errors and biases in sequencing technologies, and the large volumes of sequencing data required for metagenome sequencing has led to unacceptably high false discovery rates (FDR). Here, we present the application of a novel, gene-independent and signature-based metagenomic taxonomic profiling method with significantly and consistently smaller FDR than any other available method. Our algorithm circumvents false positives using a series of non-redundant signature databases and examines Genomic Origins Through Taxonomic CHAllenge (GOTTCHA). GOTTCHA was tested and validated on 20 synthetic and mock datasets ranging in community composition and complexity, was applied successfully to data generated from spiked environmental and clinical samples, and robustly demonstrates superior performance compared with other available tools. PMID:25765641

  20. Experimental Influences in the Accurate Measurement of Cartilage Thickness in MRI.

    PubMed

    Wang, Nian; Badar, Farid; Xia, Yang

    2018-01-01

    Objective To study the experimental influences to the measurement of cartilage thickness by magnetic resonance imaging (MRI). Design The complete thicknesses of healthy and trypsin-degraded cartilage were measured at high-resolution MRI under different conditions, using two intensity-based imaging sequences (ultra-short echo [UTE] and multislice-multiecho [MSME]) and 3 quantitative relaxation imaging sequences (T 1 , T 2 , and T 1 ρ). Other variables included different orientations in the magnet, 2 soaking solutions (saline and phosphate buffered saline [PBS]), and external loading. Results With cartilage soaked in saline, UTE and T 1 methods yielded complete and consistent measurement of cartilage thickness, while the thickness measurement by T 2 , T 1 ρ, and MSME methods were orientation dependent. The effect of external loading on cartilage thickness is also sequence and orientation dependent. All variations in cartilage thickness in MRI could be eliminated with the use of a 100 mM PBS or imaged by UTE sequence. Conclusions The appearance of articular cartilage and the measurement accuracy of cartilage thickness in MRI can be influenced by a number of experimental factors in ex vivo MRI, from the use of various pulse sequences and soaking solutions to the health of the tissue. T 2 -based imaging sequence, both proton-intensity sequence and quantitative relaxation sequence, similarly produced the largest variations. With adequate resolution, the accurate measurement of whole cartilage tissue in clinical MRI could be utilized to detect differences between healthy and osteoarthritic cartilage after compression.

  1. ChIP-seq Accurately Predicts Tissue-Specific Activity of Enhancers

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

    Visel, Axel; Blow, Matthew J.; Li, Zirong

    2009-02-01

    A major yet unresolved quest in decoding the human genome is the identification of the regulatory sequences that control the spatial and temporal expression of genes. Distant-acting transcriptional enhancers are particularly challenging to uncover since they are scattered amongst the vast non-coding portion of the genome. Evolutionary sequence constraint can facilitate the discovery of enhancers, but fails to predict when and where they are active in vivo. Here, we performed chromatin immunoprecipitation with the enhancer-associated protein p300, followed by massively-parallel sequencing, to map several thousand in vivo binding sites of p300 in mouse embryonic forebrain, midbrain, and limb tissue. Wemore » tested 86 of these sequences in a transgenic mouse assay, which in nearly all cases revealed reproducible enhancer activity in those tissues predicted by p300 binding. Our results indicate that in vivo mapping of p300 binding is a highly accurate means for identifying enhancers and their associated activities and suggest that such datasets will be useful to study the role of tissue-specific enhancers in human biology and disease on a genome-wide scale.« less

  2. Single-Cell RNA-Sequencing in Glioma.

    PubMed

    Johnson, Eli; Dickerson, Katherine L; Connolly, Ian D; Hayden Gephart, Melanie

    2018-04-10

    In this review, we seek to summarize the literature concerning the use of single-cell RNA-sequencing for CNS gliomas. Single-cell analysis has revealed complex tumor heterogeneity, subpopulations of proliferating stem-like cells and expanded our view of tumor microenvironment influence in the disease process. Although bulk RNA-sequencing has guided our initial understanding of glioma genetics, this method does not accurately define the heterogeneous subpopulations found within these tumors. Single-cell techniques have appealing applications in cancer research, as diverse cell types and the tumor microenvironment have important implications in therapy. High cost and difficult protocols prevent widespread use of single-cell RNA-sequencing; however, continued innovation will improve accessibility and expand our of knowledge gliomas.

  3. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

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

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonucleasemore » I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.« less

  4. Accurate, Rapid Taxonomic Classification of Fungal Large-Subunit rRNA Genes

    PubMed Central

    Liu, Kuan-Liang; Porras-Alfaro, Andrea; Eichorst, Stephanie A.

    2012-01-01

    Taxonomic and phylogenetic fingerprinting based on sequence analysis of gene fragments from the large-subunit rRNA (LSU) gene or the internal transcribed spacer (ITS) region is becoming an integral part of fungal classification. The lack of an accurate and robust classification tool trained by a validated sequence database for taxonomic placement of fungal LSU genes is a severe limitation in taxonomic analysis of fungal isolates or large data sets obtained from environmental surveys. Using a hand-curated set of 8,506 fungal LSU gene fragments, we determined the performance characteristics of a naïve Bayesian classifier across multiple taxonomic levels and compared the classifier performance to that of a sequence similarity-based (BLASTN) approach. The naïve Bayesian classifier was computationally more rapid (>460-fold with our system) than the BLASTN approach, and it provided equal or superior classification accuracy. Classifier accuracies were compared using sequence fragments of 100 bp and 400 bp and two different PCR primer anchor points to mimic sequence read lengths commonly obtained using current high-throughput sequencing technologies. Accuracy was higher with 400-bp sequence reads than with 100-bp reads. It was also significantly affected by sequence location across the 1,400-bp test region. The highest accuracy was obtained across either the D1 or D2 variable region. The naïve Bayesian classifier provides an effective and rapid means to classify fungal LSU sequences from large environmental surveys. The training set and tool are publicly available through the Ribosomal Database Project (http://rdp.cme.msu.edu/classifier/classifier.jsp). PMID:22194300

  5. FRAGS: estimation of coding sequence substitution rates from fragmentary data

    PubMed Central

    Swart, Estienne C; Hide, Winston A; Seoighe, Cathal

    2004-01-01

    Background Rates of substitution in protein-coding sequences can provide important insights into evolutionary processes that are of biomedical and theoretical interest. Increased availability of coding sequence data has enabled researchers to estimate more accurately the coding sequence divergence of pairs of organisms. However the use of different data sources, alignment protocols and methods to estimate substitution rates leads to widely varying estimates of key parameters that define the coding sequence divergence of orthologous genes. Although complete genome sequence data are not available for all organisms, fragmentary sequence data can provide accurate estimates of substitution rates provided that an appropriate and consistent methodology is used and that differences in the estimates obtainable from different data sources are taken into account. Results We have developed FRAGS, an application framework that uses existing, freely available software components to construct in-frame alignments and estimate coding substitution rates from fragmentary sequence data. Coding sequence substitution estimates for human and chimpanzee sequences, generated by FRAGS, reveal that methodological differences can give rise to significantly different estimates of important substitution parameters. The estimated substitution rates were also used to infer upper-bounds on the amount of sequencing error in the datasets that we have analysed. Conclusion We have developed a system that performs robust estimation of substitution rates for orthologous sequences from a pair of organisms. Our system can be used when fragmentary genomic or transcript data is available from one of the organisms and the other is a completely sequenced genome within the Ensembl database. As well as estimating substitution statistics our system enables the user to manage and query alignment and substitution data. PMID:15005802

  6. Successful amplification of DNA aboard the International Space Station.

    PubMed

    Boguraev, Anna-Sophia; Christensen, Holly C; Bonneau, Ashley R; Pezza, John A; Nichols, Nicole M; Giraldez, Antonio J; Gray, Michelle M; Wagner, Brandon M; Aken, Jordan T; Foley, Kevin D; Copeland, D Scott; Kraves, Sebastian; Alvarez Saavedra, Ezequiel

    2017-01-01

    As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is the gold standard for DNA analysis, yet its potential for use on-orbit remains under-explored. We describe DNA amplification aboard the International Space Station (ISS) through the use of a miniaturized miniPCR system. Target sequences in plasmid, zebrafish genomic DNA, and bisulfite-treated DNA were successfully amplified under a variety of conditions. Methylation-specific primers differentially amplified bisulfite-treated samples as would be expected under standard laboratory conditions. Our findings establish proof of concept for targeted detection of DNA sequences during spaceflight and lay a foundation for future uses ranging from environmental monitoring to on-orbit diagnostics.

  7. Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tumor tissue-of-origin mapping from plasma DNA.

    PubMed

    Guo, Shicheng; Diep, Dinh; Plongthongkum, Nongluk; Fung, Ho-Lim; Zhang, Kang; Zhang, Kun

    2017-04-01

    Adjacent CpG sites in mammalian genomes can be co-methylated owing to the processivity of methyltransferases or demethylases, yet discordant methylation patterns have also been observed, which are related to stochastic or uncoordinated molecular processes. We focused on a systematic search and investigation of regions in the full human genome that show highly coordinated methylation. We defined 147,888 blocks of tightly coupled CpG sites, called methylation haplotype blocks, after analysis of 61 whole-genome bisulfite sequencing data sets and validation with 101 reduced-representation bisulfite sequencing data sets and 637 methylation array data sets. Using a metric called methylation haplotype load, we performed tissue-specific methylation analysis at the block level. Subsets of informative blocks were further identified for deconvolution of heterogeneous samples. Finally, using methylation haplotypes we demonstrated quantitative estimation of tumor load and tissue-of-origin mapping in the circulating cell-free DNA of 59 patients with lung or colorectal cancer.

  8. Mycofier: a new machine learning-based classifier for fungal ITS sequences.

    PubMed

    Delgado-Serrano, Luisa; Restrepo, Silvia; Bustos, Jose Ricardo; Zambrano, Maria Mercedes; Anzola, Juan Manuel

    2016-08-11

    The taxonomic and phylogenetic classification based on sequence analysis of the ITS1 genomic region has become a crucial component of fungal ecology and diversity studies. Nowadays, there is no accurate alignment-free classification tool for fungal ITS1 sequences for large environmental surveys. This study describes the development of a machine learning-based classifier for the taxonomical assignment of fungal ITS1 sequences at the genus level. A fungal ITS1 sequence database was built using curated data. Training and test sets were generated from it. A Naïve Bayesian classifier was built using features from the primary sequence with an accuracy of 87 % in the classification at the genus level. The final model was based on a Naïve Bayes algorithm using ITS1 sequences from 510 fungal genera. This classifier, denoted as Mycofier, provides similar classification accuracy compared to BLASTN, but the database used for the classification contains curated data and the tool, independent of alignment, is more efficient and contributes to the field, given the lack of an accurate classification tool for large data from fungal ITS1 sequences. The software and source code for Mycofier are freely available at https://github.com/ldelgado-serrano/mycofier.git .

  9. DNA Barcode Sequence Identification Incorporating Taxonomic Hierarchy and within Taxon Variability

    PubMed Central

    Little, Damon P.

    2011-01-01

    For DNA barcoding to succeed as a scientific endeavor an accurate and expeditious query sequence identification method is needed. Although a global multiple–sequence alignment can be generated for some barcoding markers (e.g. COI, rbcL), not all barcoding markers are as structurally conserved (e.g. matK). Thus, algorithms that depend on global multiple–sequence alignments are not universally applicable. Some sequence identification methods that use local pairwise alignments (e.g. BLAST) are unable to accurately differentiate between highly similar sequences and are not designed to cope with hierarchic phylogenetic relationships or within taxon variability. Here, I present a novel alignment–free sequence identification algorithm–BRONX–that accounts for observed within taxon variability and hierarchic relationships among taxa. BRONX identifies short variable segments and corresponding invariant flanking regions in reference sequences. These flanking regions are used to score variable regions in the query sequence without the production of a global multiple–sequence alignment. By incorporating observed within taxon variability into the scoring procedure, misidentifications arising from shared alleles/haplotypes are minimized. An explicit treatment of more inclusive terminals allows for separate identifications to be made for each taxonomic level and/or for user–defined terminals. BRONX performs better than all other methods when there is imperfect overlap between query and reference sequences (e.g. mini–barcode queries against a full–length barcode database). BRONX consistently produced better identifications at the genus–level for all query types. PMID:21857897

  10. A Dual-Mode Large-Arrayed CMOS ISFET Sensor for Accurate and High-Throughput pH Sensing in Biomedical Diagnosis.

    PubMed

    Huang, Xiwei; Yu, Hao; Liu, Xu; Jiang, Yu; Yan, Mei; Wu, Dongping

    2015-09-01

    The existing ISFET-based DNA sequencing detects hydrogen ions released during the polymerization of DNA strands on microbeads, which are scattered into microwell array above the ISFET sensor with unknown distribution. However, false pH detection happens at empty microwells due to crosstalk from neighboring microbeads. In this paper, a dual-mode CMOS ISFET sensor is proposed to have accurate pH detection toward DNA sequencing. Dual-mode sensing, optical and chemical modes, is realized by integrating a CMOS image sensor (CIS) with ISFET pH sensor, and is fabricated in a standard 0.18-μm CIS process. With accurate determination of microbead physical locations with CIS pixel by contact imaging, the dual-mode sensor can correlate local pH for one DNA slice at one location-determined microbead, which can result in improved pH detection accuracy. Moreover, toward a high-throughput DNA sequencing, a correlated-double-sampling readout that supports large array for both modes is deployed to reduce pixel-to-pixel nonuniformity such as threshold voltage mismatch. The proposed CMOS dual-mode sensor is experimentally examined to show a well correlated pH map and optical image for microbeads with a pH sensitivity of 26.2 mV/pH, a fixed pattern noise (FPN) reduction from 4% to 0.3%, and a readout speed of 1200 frames/s. A dual-mode CMOS ISFET sensor with suppressed FPN for accurate large-arrayed pH sensing is proposed and demonstrated with state-of-the-art measured results toward accurate and high-throughput DNA sequencing. The developed dual-mode CMOS ISFET sensor has great potential for future personal genome diagnostics with high accuracy and low cost.

  11. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.

    PubMed

    Chin, Chen-Shan; Alexander, David H; Marks, Patrick; Klammer, Aaron A; Drake, James; Heiner, Cheryl; Clum, Alicia; Copeland, Alex; Huddleston, John; Eichler, Evan E; Turner, Stephen W; Korlach, Jonas

    2013-06-01

    We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.

  12. PSI/TM-Coffee: a web server for fast and accurate multiple sequence alignments of regular and transmembrane proteins using homology extension on reduced databases.

    PubMed

    Floden, Evan W; Tommaso, Paolo D; Chatzou, Maria; Magis, Cedrik; Notredame, Cedric; Chang, Jia-Ming

    2016-07-08

    The PSI/TM-Coffee web server performs multiple sequence alignment (MSA) of proteins by combining homology extension with a consistency based alignment approach. Homology extension is performed with Position Specific Iterative (PSI) BLAST searches against a choice of redundant and non-redundant databases. The main novelty of this server is to allow databases of reduced complexity to rapidly perform homology extension. This server also gives the possibility to use transmembrane proteins (TMPs) reference databases to allow even faster homology extension on this important category of proteins. Aside from an MSA, the server also outputs topological prediction of TMPs using the HMMTOP algorithm. Previous benchmarking of the method has shown this approach outperforms the most accurate alignment methods such as MSAProbs, Kalign, PROMALS, MAFFT, ProbCons and PRALINE™. The web server is available at http://tcoffee.crg.cat/tmcoffee. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Centrifuge: rapid and sensitive classification of metagenomic sequences.

    PubMed

    Kim, Daehwan; Song, Li; Breitwieser, Florian P; Salzberg, Steven L

    2016-12-01

    Centrifuge is a novel microbial classification engine that enables rapid, accurate, and sensitive labeling of reads and quantification of species on desktop computers. The system uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index, optimized specifically for the metagenomic classification problem. Centrifuge requires a relatively small index (4.2 GB for 4078 bacterial and 200 archaeal genomes) and classifies sequences at very high speed, allowing it to process the millions of reads from a typical high-throughput DNA sequencing run within a few minutes. Together, these advances enable timely and accurate analysis of large metagenomics data sets on conventional desktop computers. Because of its space-optimized indexing schemes, Centrifuge also makes it possible to index the entire NCBI nonredundant nucleotide sequence database (a total of 109 billion bases) with an index size of 69 GB, in contrast to k-mer-based indexing schemes, which require far more extensive space. © 2016 Kim et al.; Published by Cold Spring Harbor Laboratory Press.

  14. PredPPCrys: Accurate Prediction of Sequence Cloning, Protein Production, Purification and Crystallization Propensity from Protein Sequences Using Multi-Step Heterogeneous Feature Fusion and Selection

    PubMed Central

    Wang, Huilin; Wang, Mingjun; Tan, Hao; Li, Yuan; Zhang, Ziding; Song, Jiangning

    2014-01-01

    X-ray crystallography is the primary approach to solve the three-dimensional structure of a protein. However, a major bottleneck of this method is the failure of multi-step experimental procedures to yield diffraction-quality crystals, including sequence cloning, protein material production, purification, crystallization and ultimately, structural determination. Accordingly, prediction of the propensity of a protein to successfully undergo these experimental procedures based on the protein sequence may help narrow down laborious experimental efforts and facilitate target selection. A number of bioinformatics methods based on protein sequence information have been developed for this purpose. However, our knowledge on the important determinants of propensity for a protein sequence to produce high diffraction-quality crystals remains largely incomplete. In practice, most of the existing methods display poorer performance when evaluated on larger and updated datasets. To address this problem, we constructed an up-to-date dataset as the benchmark, and subsequently developed a new approach termed ‘PredPPCrys’ using the support vector machine (SVM). Using a comprehensive set of multifaceted sequence-derived features in combination with a novel multi-step feature selection strategy, we identified and characterized the relative importance and contribution of each feature type to the prediction performance of five individual experimental steps required for successful crystallization. The resulting optimal candidate features were used as inputs to build the first-level SVM predictor (PredPPCrys I). Next, prediction outputs of PredPPCrys I were used as the input to build second-level SVM classifiers (PredPPCrys II), which led to significantly enhanced prediction performance. Benchmarking experiments indicated that our PredPPCrys method outperforms most existing procedures on both up-to-date and previous datasets. In addition, the predicted crystallization targets of

  15. PredPPCrys: accurate prediction of sequence cloning, protein production, purification and crystallization propensity from protein sequences using multi-step heterogeneous feature fusion and selection.

    PubMed

    Wang, Huilin; Wang, Mingjun; Tan, Hao; Li, Yuan; Zhang, Ziding; Song, Jiangning

    2014-01-01

    X-ray crystallography is the primary approach to solve the three-dimensional structure of a protein. However, a major bottleneck of this method is the failure of multi-step experimental procedures to yield diffraction-quality crystals, including sequence cloning, protein material production, purification, crystallization and ultimately, structural determination. Accordingly, prediction of the propensity of a protein to successfully undergo these experimental procedures based on the protein sequence may help narrow down laborious experimental efforts and facilitate target selection. A number of bioinformatics methods based on protein sequence information have been developed for this purpose. However, our knowledge on the important determinants of propensity for a protein sequence to produce high diffraction-quality crystals remains largely incomplete. In practice, most of the existing methods display poorer performance when evaluated on larger and updated datasets. To address this problem, we constructed an up-to-date dataset as the benchmark, and subsequently developed a new approach termed 'PredPPCrys' using the support vector machine (SVM). Using a comprehensive set of multifaceted sequence-derived features in combination with a novel multi-step feature selection strategy, we identified and characterized the relative importance and contribution of each feature type to the prediction performance of five individual experimental steps required for successful crystallization. The resulting optimal candidate features were used as inputs to build the first-level SVM predictor (PredPPCrys I). Next, prediction outputs of PredPPCrys I were used as the input to build second-level SVM classifiers (PredPPCrys II), which led to significantly enhanced prediction performance. Benchmarking experiments indicated that our PredPPCrys method outperforms most existing procedures on both up-to-date and previous datasets. In addition, the predicted crystallization targets of currently

  16. NullSeq: A Tool for Generating Random Coding Sequences with Desired Amino Acid and GC Contents.

    PubMed

    Liu, Sophia S; Hockenberry, Adam J; Lancichinetti, Andrea; Jewett, Michael C; Amaral, Luís A N

    2016-11-01

    The existence of over- and under-represented sequence motifs in genomes provides evidence of selective evolutionary pressures on biological mechanisms such as transcription, translation, ligand-substrate binding, and host immunity. In order to accurately identify motifs and other genome-scale patterns of interest, it is essential to be able to generate accurate null models that are appropriate for the sequences under study. While many tools have been developed to create random nucleotide sequences, protein coding sequences are subject to a unique set of constraints that complicates the process of generating appropriate null models. There are currently no tools available that allow users to create random coding sequences with specified amino acid composition and GC content for the purpose of hypothesis testing. Using the principle of maximum entropy, we developed a method that generates unbiased random sequences with pre-specified amino acid and GC content, which we have developed into a python package. Our method is the simplest way to obtain maximally unbiased random sequences that are subject to GC usage and primary amino acid sequence constraints. Furthermore, this approach can easily be expanded to create unbiased random sequences that incorporate more complicated constraints such as individual nucleotide usage or even di-nucleotide frequencies. The ability to generate correctly specified null models will allow researchers to accurately identify sequence motifs which will lead to a better understanding of biological processes as well as more effective engineering of biological systems.

  17. miRCat2: accurate prediction of plant and animal microRNAs from next-generation sequencing datasets

    PubMed Central

    Paicu, Claudia; Mohorianu, Irina; Stocks, Matthew; Xu, Ping; Coince, Aurore; Billmeier, Martina; Dalmay, Tamas; Moulton, Vincent; Moxon, Simon

    2017-01-01

    Abstract Motivation MicroRNAs are a class of ∼21–22 nt small RNAs which are excised from a stable hairpin-like secondary structure. They have important gene regulatory functions and are involved in many pathways including developmental timing, organogenesis and development in eukaryotes. There are several computational tools for miRNA detection from next-generation sequencing datasets. However, many of these tools suffer from high false positive and false negative rates. Here we present a novel miRNA prediction algorithm, miRCat2. miRCat2 incorporates a new entropy-based approach to detect miRNA loci, which is designed to cope with the high sequencing depth of current next-generation sequencing datasets. It has a user-friendly interface and produces graphical representations of the hairpin structure and plots depicting the alignment of sequences on the secondary structure. Results We test miRCat2 on a number of animal and plant datasets and present a comparative analysis with miRCat, miRDeep2, miRPlant and miReap. We also use mutants in the miRNA biogenesis pathway to evaluate the predictions of these tools. Results indicate that miRCat2 has an improved accuracy compared with other methods tested. Moreover, miRCat2 predicts several new miRNAs that are differentially expressed in wild-type versus mutants in the miRNA biogenesis pathway. Availability and Implementation miRCat2 is part of the UEA small RNA Workbench and is freely available from http://srna-workbench.cmp.uea.ac.uk/. Contact v.moulton@uea.ac.uk or s.moxon@uea.ac.uk Supplementary information Supplementary data are available at Bioinformatics online. PMID:28407097

  18. RCK: accurate and efficient inference of sequence- and structure-based protein-RNA binding models from RNAcompete data.

    PubMed

    Orenstein, Yaron; Wang, Yuhao; Berger, Bonnie

    2016-06-15

    Protein-RNA interactions, which play vital roles in many processes, are mediated through both RNA sequence and structure. CLIP-based methods, which measure protein-RNA binding in vivo, suffer from experimental noise and systematic biases, whereas in vitro experiments capture a clearer signal of protein RNA-binding. Among them, RNAcompete provides binding affinities of a specific protein to more than 240 000 unstructured RNA probes in one experiment. The computational challenge is to infer RNA structure- and sequence-based binding models from these data. The state-of-the-art in sequence models, Deepbind, does not model structural preferences. RNAcontext models both sequence and structure preferences, but is outperformed by GraphProt. Unfortunately, GraphProt cannot detect structural preferences from RNAcompete data due to the unstructured nature of the data, as noted by its developers, nor can it be tractably run on the full RNACompete dataset. We develop RCK, an efficient, scalable algorithm that infers both sequence and structure preferences based on a new k-mer based model. Remarkably, even though RNAcompete data is designed to be unstructured, RCK can still learn structural preferences from it. RCK significantly outperforms both RNAcontext and Deepbind in in vitro binding prediction for 244 RNAcompete experiments. Moreover, RCK is also faster and uses less memory, which enables scalability. While currently on par with existing methods in in vivo binding prediction on a small scale test, we demonstrate that RCK will increasingly benefit from experimentally measured RNA structure profiles as compared to computationally predicted ones. By running RCK on the entire RNAcompete dataset, we generate and provide as a resource a set of protein-RNA structure-based models on an unprecedented scale. Software and models are freely available at http://rck.csail.mit.edu/ bab@mit.edu Supplementary data are available at Bioinformatics online. © The Author 2016. Published by

  19. MICCA: a complete and accurate software for taxonomic profiling of metagenomic data.

    PubMed

    Albanese, Davide; Fontana, Paolo; De Filippo, Carlotta; Cavalieri, Duccio; Donati, Claudio

    2015-05-19

    The introduction of high throughput sequencing technologies has triggered an increase of the number of studies in which the microbiota of environmental and human samples is characterized through the sequencing of selected marker genes. While experimental protocols have undergone a process of standardization that makes them accessible to a large community of scientist, standard and robust data analysis pipelines are still lacking. Here we introduce MICCA, a software pipeline for the processing of amplicon metagenomic datasets that efficiently combines quality filtering, clustering of Operational Taxonomic Units (OTUs), taxonomy assignment and phylogenetic tree inference. MICCA provides accurate results reaching a good compromise among modularity and usability. Moreover, we introduce a de-novo clustering algorithm specifically designed for the inference of Operational Taxonomic Units (OTUs). Tests on real and synthetic datasets shows that thanks to the optimized reads filtering process and to the new clustering algorithm, MICCA provides estimates of the number of OTUs and of other common ecological indices that are more accurate and robust than currently available pipelines. Analysis of public metagenomic datasets shows that the higher consistency of results improves our understanding of the structure of environmental and human associated microbial communities. MICCA is an open source project.

  20. BEST: Improved Prediction of B-Cell Epitopes from Antigen Sequences

    PubMed Central

    Gao, Jianzhao; Faraggi, Eshel; Zhou, Yaoqi; Ruan, Jishou; Kurgan, Lukasz

    2012-01-01

    Accurate identification of immunogenic regions in a given antigen chain is a difficult and actively pursued problem. Although accurate predictors for T-cell epitopes are already in place, the prediction of the B-cell epitopes requires further research. We overview the available approaches for the prediction of B-cell epitopes and propose a novel and accurate sequence-based solution. Our BEST (B-cell Epitope prediction using Support vector machine Tool) method predicts epitopes from antigen sequences, in contrast to some method that predict only from short sequence fragments, using a new architecture based on averaging selected scores generated from sliding 20-mers by a Support Vector Machine (SVM). The SVM predictor utilizes a comprehensive and custom designed set of inputs generated by combining information derived from the chain, sequence conservation, similarity to known (training) epitopes, and predicted secondary structure and relative solvent accessibility. Empirical evaluation on benchmark datasets demonstrates that BEST outperforms several modern sequence-based B-cell epitope predictors including ABCPred, method by Chen et al. (2007), BCPred, COBEpro, BayesB, and CBTOPE, when considering the predictions from antigen chains and from the chain fragments. Our method obtains a cross-validated area under the receiver operating characteristic curve (AUC) for the fragment-based prediction at 0.81 and 0.85, depending on the dataset. The AUCs of BEST on the benchmark sets of full antigen chains equal 0.57 and 0.6, which is significantly and slightly better than the next best method we tested. We also present case studies to contrast the propensity profiles generated by BEST and several other methods. PMID:22761950

  1. Nonparametric Bayesian clustering to detect bipolar methylated genomic loci.

    PubMed

    Wu, Xiaowei; Sun, Ming-An; Zhu, Hongxiao; Xie, Hehuang

    2015-01-16

    With recent development in sequencing technology, a large number of genome-wide DNA methylation studies have generated massive amounts of bisulfite sequencing data. The analysis of DNA methylation patterns helps researchers understand epigenetic regulatory mechanisms. Highly variable methylation patterns reflect stochastic fluctuations in DNA methylation, whereas well-structured methylation patterns imply deterministic methylation events. Among these methylation patterns, bipolar patterns are important as they may originate from allele-specific methylation (ASM) or cell-specific methylation (CSM). Utilizing nonparametric Bayesian clustering followed by hypothesis testing, we have developed a novel statistical approach to identify bipolar methylated genomic regions in bisulfite sequencing data. Simulation studies demonstrate that the proposed method achieves good performance in terms of specificity and sensitivity. We used the method to analyze data from mouse brain and human blood methylomes. The bipolar methylated segments detected are found highly consistent with the differentially methylated regions identified by using purified cell subsets. Bipolar DNA methylation often indicates epigenetic heterogeneity caused by ASM or CSM. With allele-specific events filtered out or appropriately taken into account, our proposed approach sheds light on the identification of cell-specific genes/pathways under strong epigenetic control in a heterogeneous cell population.

  2. A novel approach to multiple sequence alignment using hadoop data grids.

    PubMed

    Sudha Sadasivam, G; Baktavatchalam, G

    2010-01-01

    Multiple alignment of protein sequences helps to determine evolutionary linkage and to predict molecular structures. The factors to be considered while aligning multiple sequences are speed and accuracy of alignment. Although dynamic programming algorithms produce accurate alignments, they are computation intensive. In this paper we propose a time efficient approach to sequence alignment that also produces quality alignment. The dynamic nature of the algorithm coupled with data and computational parallelism of hadoop data grids improves the accuracy and speed of sequence alignment. The principle of block splitting in hadoop coupled with its scalability facilitates alignment of very large sequences.

  3. Transforming clinical microbiology with bacterial genome sequencing.

    PubMed

    Didelot, Xavier; Bowden, Rory; Wilson, Daniel J; Peto, Tim E A; Crook, Derrick W

    2012-09-01

    Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for addressing many microbiological questions. Here, we review the current status of clinical microbiology and how it has already begun to be transformed by using next-generation sequencing. We focus on three essential tasks: identifying the species of an isolate, testing its properties, such as resistance to antibiotics and virulence, and monitoring the emergence and spread of bacterial pathogens. We predict that the application of next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice, where it could replace many complex current techniques with a single, more efficient workflow.

  4. Transforming clinical microbiology with bacterial genome sequencing

    PubMed Central

    2016-01-01

    Whole genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for addressing many microbiological questions. Here we review the current status of clinical microbiology and how it has already begun to be transformed by the use of next-generation sequencing. We focus on three essential tasks: identifying the species of an isolate, testing its properties such as resistance to antibiotics and virulence, and monitoring the emergence and spread of bacterial pathogens. The application of next-generation sequencing will soon be sufficiently fast, accurate and cheap to be used in routine clinical microbiology practice, where it could replace many complex current techniques with a single, more efficient workflow. PMID:22868263

  5. Ancestry estimation and control of population stratification for sequence-based association studies.

    PubMed

    Wang, Chaolong; Zhan, Xiaowei; Bragg-Gresham, Jennifer; Kang, Hyun Min; Stambolian, Dwight; Chew, Emily Y; Branham, Kari E; Heckenlively, John; Fulton, Robert; Wilson, Richard K; Mardis, Elaine R; Lin, Xihong; Swaroop, Anand; Zöllner, Sebastian; Abecasis, Gonçalo R

    2014-04-01

    Estimating individual ancestry is important in genetic association studies where population structure leads to false positive signals, although assigning ancestry remains challenging with targeted sequence data. We propose a new method for the accurate estimation of individual genetic ancestry, based on direct analysis of off-target sequence reads, and implement our method in the publicly available LASER software. We validate the method using simulated and empirical data and show that the method can accurately infer worldwide continental ancestry when used with sequencing data sets with whole-genome shotgun coverage as low as 0.001×. For estimates of fine-scale ancestry within Europe, the method performs well with coverage of 0.1×. On an even finer scale, the method improves discrimination between exome-sequenced study participants originating from different provinces within Finland. Finally, we show that our method can be used to improve case-control matching in genetic association studies and to reduce the risk of spurious findings due to population structure.

  6. A Probabilistic Model of Local Sequence Alignment That Simplifies Statistical Significance Estimation

    PubMed Central

    Eddy, Sean R.

    2008-01-01

    Sequence database searches require accurate estimation of the statistical significance of scores. Optimal local sequence alignment scores follow Gumbel distributions, but determining an important parameter of the distribution (λ) requires time-consuming computational simulation. Moreover, optimal alignment scores are less powerful than probabilistic scores that integrate over alignment uncertainty (“Forward” scores), but the expected distribution of Forward scores remains unknown. Here, I conjecture that both expected score distributions have simple, predictable forms when full probabilistic modeling methods are used. For a probabilistic model of local sequence alignment, optimal alignment bit scores (“Viterbi” scores) are Gumbel-distributed with constant λ = log 2, and the high scoring tail of Forward scores is exponential with the same constant λ. Simulation studies support these conjectures over a wide range of profile/sequence comparisons, using 9,318 profile-hidden Markov models from the Pfam database. This enables efficient and accurate determination of expectation values (E-values) for both Viterbi and Forward scores for probabilistic local alignments. PMID:18516236

  7. The Influence of Hydroxylation on Maintaining CpG Methylation Patterns: A Hidden Markov Model Approach.

    PubMed

    Giehr, Pascal; Kyriakopoulos, Charalampos; Ficz, Gabriella; Wolf, Verena; Walter, Jörn

    2016-05-01

    DNA methylation and demethylation are opposing processes that when in balance create stable patterns of epigenetic memory. The control of DNA methylation pattern formation by replication dependent and independent demethylation processes has been suggested to be influenced by Tet mediated oxidation of 5mC. Several alternative mechanisms have been proposed suggesting that 5hmC influences either replication dependent maintenance of DNA methylation or replication independent processes of active demethylation. Using high resolution hairpin oxidative bisulfite sequencing data, we precisely determine the amount of 5mC and 5hmC and model the contribution of 5hmC to processes of demethylation in mouse ESCs. We develop an extended hidden Markov model capable of accurately describing the regional contribution of 5hmC to demethylation dynamics. Our analysis shows that 5hmC has a strong impact on replication dependent demethylation, mainly by impairing methylation maintenance.

  8. Accurate high-speed liquid handling of very small biological samples.

    PubMed

    Schober, A; Günther, R; Schwienhorst, A; Döring, M; Lindemann, B F

    1993-08-01

    Molecular biology techniques require the accurate pipetting of buffers and solutions with volumes in the microliter range. Traditionally, hand-held pipetting devices are used to fulfill these requirements, but many laboratories have also introduced robotic workstations for the handling of liquids. Piston-operated pumps are commonly used in manually as well as automatically operated pipettors. These devices cannot meet the demands for extremely accurate pipetting of very small volumes at the high speed that would be necessary for certain applications (e.g., in sequencing projects with high throughput). In this paper we describe a technique for the accurate microdispensation of biochemically relevant solutions and suspensions with the aid of a piezoelectric transducer. It is suitable for liquids of a viscosity between 0.5 and 500 milliPascals. The obtainable drop sizes range from 5 picoliters to a few nanoliters with up to 10,000 drops per second. Liquids can be dispensed in single or accumulated drops to handle a wide volume range. The system proved to be excellently suitable for the handling of biological samples. It did not show any detectable negative impact on the biological function of dissolved or suspended molecules or particles.

  9. Sequencing Cyclic Peptides by Multistage Mass Spectrometry

    PubMed Central

    Mohimani, Hosein; Yang, Yu-Liang; Liu, Wei-Ting; Hsieh, Pei-Wen; Dorrestein, Pieter C.; Pevzner, Pavel A.

    2012-01-01

    Some of the most effective antibiotics (e.g., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of mass spectrometry based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage mass spectrometry, and show its advantages over single stage mass spectrometry. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria. PMID:21751357

  10. Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

    PubMed Central

    Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

    2013-01-01

    For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

  11. Combinatorial pooling enables selective sequencing of the barley gene space.

    PubMed

    Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J

    2013-04-01

    For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

  12. Independent assessment and improvement of wheat genome sequence assemblies using Fosill jumping libraries.

    PubMed

    Lu, Fu-Hao; McKenzie, Neil; Kettleborough, George; Heavens, Darren; Clark, Matthew D; Bevan, Michael W

    2018-05-01

    The accurate sequencing and assembly of very large, often polyploid, genomes remains a challenging task, limiting long-range sequence information and phased sequence variation for applications such as plant breeding. The 15-Gb hexaploid bread wheat (Triticum aestivum) genome has been particularly challenging to sequence, and several different approaches have recently generated long-range assemblies. Mapping and understanding the types of assembly errors are important for optimising future sequencing and assembly approaches and for comparative genomics. Here we use a Fosill 38-kb jumping library to assess medium and longer-range order of different publicly available wheat genome assemblies. Modifications to the Fosill protocol generated longer Illumina sequences and enabled comprehensive genome coverage. Analyses of two independent Bacterial Artificial Chromosome (BAC)-based chromosome-scale assemblies, two independent Illumina whole genome shotgun assemblies, and a hybrid Single Molecule Real Time (SMRT-PacBio) and short read (Illumina) assembly were carried out. We revealed a surprising scale and variety of discrepancies using Fosill mate-pair mapping and validated several of each class. In addition, Fosill mate-pairs were used to scaffold a whole genome Illumina assembly, leading to a 3-fold increase in N50 values. Our analyses, using an independent means to validate different wheat genome assemblies, show that whole genome shotgun assemblies based solely on Illumina sequences are significantly more accurate by all measures compared to BAC-based chromosome-scale assemblies and hybrid SMRT-Illumina approaches. Although current whole genome assemblies are reasonably accurate and useful, additional improvements will be needed to generate complete assemblies of wheat genomes using open-source, computationally efficient, and cost-effective methods.

  13. Uniform, optimal signal processing of mapped deep-sequencing data.

    PubMed

    Kumar, Vibhor; Muratani, Masafumi; Rayan, Nirmala Arul; Kraus, Petra; Lufkin, Thomas; Ng, Huck Hui; Prabhakar, Shyam

    2013-07-01

    Despite their apparent diversity, many problems in the analysis of high-throughput sequencing data are merely special cases of two general problems, signal detection and signal estimation. Here we adapt formally optimal solutions from signal processing theory to analyze signals of DNA sequence reads mapped to a genome. We describe DFilter, a detection algorithm that identifies regulatory features in ChIP-seq, DNase-seq and FAIRE-seq data more accurately than assay-specific algorithms. We also describe EFilter, an estimation algorithm that accurately predicts mRNA levels from as few as 1-2 histone profiles (R ∼0.9). Notably, the presence of regulatory motifs in promoters correlates more with histone modifications than with mRNA levels, suggesting that histone profiles are more predictive of cis-regulatory mechanisms. We show by applying DFilter and EFilter to embryonic forebrain ChIP-seq data that regulatory protein identification and functional annotation are feasible despite tissue heterogeneity. The mathematical formalism underlying our tools facilitates integrative analysis of data from virtually any sequencing-based functional profile.

  14. Multiplexed microsatellite recovery using massively parallel sequencing

    Treesearch

    T.N. Jennings; B.J. Knaus; T.D. Mullins; S.M. Haig; R.C. Cronn

    2011-01-01

    Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of...

  15. Shotgun Protein Sequencing with Meta-contig Assembly*

    PubMed Central

    Guthals, Adrian; Clauser, Karl R.; Bandeira, Nuno

    2012-01-01

    Full-length de novo sequencing from tandem mass (MS/MS) spectra of unknown proteins such as antibodies or proteins from organisms with unsequenced genomes remains a challenging open problem. Conventional algorithms designed to individually sequence each MS/MS spectrum are limited by incomplete peptide fragmentation or low signal to noise ratios and tend to result in short de novo sequences at low sequencing accuracy. Our shotgun protein sequencing (SPS) approach was developed to ameliorate these limitations by first finding groups of unidentified spectra from the same peptides (contigs) and then deriving a consensus de novo sequence for each assembled set of spectra (contig sequences). But whereas SPS enables much more accurate reconstruction of de novo sequences longer than can be recovered from individual MS/MS spectra, it still requires error-tolerant matching to homologous proteins to group smaller contig sequences into full-length protein sequences, thus limiting its effectiveness on sequences from poorly annotated proteins. Using low and high resolution CID and high resolution HCD MS/MS spectra, we address this limitation with a Meta-SPS algorithm designed to overlap and further assemble SPS contigs into Meta-SPS de novo contig sequences extending as long as 100 amino acids at over 97% accuracy without requiring any knowledge of homologous protein sequences. We demonstrate Meta-SPS using distinct MS/MS data sets obtained with separate enzymatic digestions and discuss how the remaining de novo sequencing limitations relate to MS/MS acquisition settings. PMID:22798278

  16. Shotgun protein sequencing with meta-contig assembly.

    PubMed

    Guthals, Adrian; Clauser, Karl R; Bandeira, Nuno

    2012-10-01

    Full-length de novo sequencing from tandem mass (MS/MS) spectra of unknown proteins such as antibodies or proteins from organisms with unsequenced genomes remains a challenging open problem. Conventional algorithms designed to individually sequence each MS/MS spectrum are limited by incomplete peptide fragmentation or low signal to noise ratios and tend to result in short de novo sequences at low sequencing accuracy. Our shotgun protein sequencing (SPS) approach was developed to ameliorate these limitations by first finding groups of unidentified spectra from the same peptides (contigs) and then deriving a consensus de novo sequence for each assembled set of spectra (contig sequences). But whereas SPS enables much more accurate reconstruction of de novo sequences longer than can be recovered from individual MS/MS spectra, it still requires error-tolerant matching to homologous proteins to group smaller contig sequences into full-length protein sequences, thus limiting its effectiveness on sequences from poorly annotated proteins. Using low and high resolution CID and high resolution HCD MS/MS spectra, we address this limitation with a Meta-SPS algorithm designed to overlap and further assemble SPS contigs into Meta-SPS de novo contig sequences extending as long as 100 amino acids at over 97% accuracy without requiring any knowledge of homologous protein sequences. We demonstrate Meta-SPS using distinct MS/MS data sets obtained with separate enzymatic digestions and discuss how the remaining de novo sequencing limitations relate to MS/MS acquisition settings.

  17. Multiplexed microsatellite recovery using massively parallel sequencing

    USGS Publications Warehouse

    Jennings, T.N.; Knaus, B.J.; Mullins, T.D.; Haig, S.M.; Cronn, R.C.

    2011-01-01

    Conservation and management of natural populations requires accurate and inexpensive genotyping methods. Traditional microsatellite, or simple sequence repeat (SSR), marker analysis remains a popular genotyping method because of the comparatively low cost of marker development, ease of analysis and high power of genotype discrimination. With the availability of massively parallel sequencing (MPS), it is now possible to sequence microsatellite-enriched genomic libraries in multiplex pools. To test this approach, we prepared seven microsatellite-enriched, barcoded genomic libraries from diverse taxa (two conifer trees, five birds) and sequenced these on one lane of the Illumina Genome Analyzer using paired-end 80-bp reads. In this experiment, we screened 6.1 million sequences and identified 356958 unique microreads that contained di- or trinucleotide microsatellites. Examination of four species shows that our conversion rate from raw sequences to polymorphic markers compares favourably to Sanger- and 454-based methods. The advantage of multiplexed MPS is that the staggering capacity of modern microread sequencing is spread across many libraries; this reduces sample preparation and sequencing costs to less than $400 (USD) per species. This price is sufficiently low that microsatellite libraries could be prepared and sequenced for all 1373 organisms listed as 'threatened' and 'endangered' in the United States for under $0.5M (USD).

  18. Illumina Synthetic Long Read Sequencing Allows Recovery of Missing Sequences even in the “Finished” C. elegans Genome

    PubMed Central

    Li, Runsheng; Hsieh, Chia-Ling; Young, Amanda; Zhang, Zhihong; Ren, Xiaoliang; Zhao, Zhongying

    2015-01-01

    Most next-generation sequencing platforms permit acquisition of high-throughput DNA sequences, but the relatively short read length limits their use in genome assembly or finishing. Illumina has recently released a technology called Synthetic Long-Read Sequencing that can produce reads of unusual length, i.e., predominately around 10 Kb. However, a systematic assessment of their use in genome finishing and assembly is still lacking. We evaluate the promise and deficiency of the long reads in these aspects using isogenic C. elegans genome with no gap. First, the reads are highly accurate and capable of recovering most types of repetitive sequences. However, the presence of tandem repetitive sequences prevents pre-assembly of long reads in the relevant genomic region. Second, the reads are able to reliably detect missing but not extra sequences in the C. elegans genome. Third, the reads of smaller size are more capable of recovering repetitive sequences than those of bigger size. Fourth, at least 40 Kbp missing genomic sequences are recovered in the C. elegans genome using the long reads. Finally, an N50 contig size of at least 86 Kbp can be achieved with 24×reads but with substantial mis-assembly errors, highlighting a need for novel assembly algorithm for the long reads. PMID:26039588

  19. Mapping DNA polymerase errors by single-molecule sequencing

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

    Lee, David F.; Lu, Jenny; Chang, Seungwoo

    Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replicationmore » product is tagged with a unique nucleotide sequence before amplification. Here, this allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases.« less

  20. Mapping DNA polymerase errors by single-molecule sequencing

    DOE PAGES

    Lee, David F.; Lu, Jenny; Chang, Seungwoo; ...

    2016-05-16

    Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replicationmore » product is tagged with a unique nucleotide sequence before amplification. Here, this allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases.« less

  1. Towards predicting the encoding capability of MR fingerprinting sequences.

    PubMed

    Sommer, K; Amthor, T; Doneva, M; Koken, P; Meineke, J; Börnert, P

    2017-09-01

    Sequence optimization and appropriate sequence selection is still an unmet need in magnetic resonance fingerprinting (MRF). The main challenge in MRF sequence design is the lack of an appropriate measure of the sequence's encoding capability. To find such a measure, three different candidates for judging the encoding capability have been investigated: local and global dot-product-based measures judging dictionary entry similarity as well as a Monte Carlo method that evaluates the noise propagation properties of an MRF sequence. Consistency of these measures for different sequence lengths as well as the capability to predict actual sequence performance in both phantom and in vivo measurements was analyzed. While the dot-product-based measures yielded inconsistent results for different sequence lengths, the Monte Carlo method was in a good agreement with phantom experiments. In particular, the Monte Carlo method could accurately predict the performance of different flip angle patterns in actual measurements. The proposed Monte Carlo method provides an appropriate measure of MRF sequence encoding capability and may be used for sequence optimization. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA

    PubMed Central

    Zill, Oliver A.; Sebisanovic, Dragan; Lopez, Rene; Blau, Sibel; Collisson, Eric A.; Divers, Stephen G.; Hoon, Dave S. B.; Kopetz, E. Scott; Lee, Jeeyun; Nikolinakos, Petros G.; Baca, Arthur M.; Kermani, Bahram G.; Eltoukhy, Helmy; Talasaz, AmirAli

    2015-01-01

    Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital SequencingTM is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient’s cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing. PMID:26474073

  3. SIFTER search: a web server for accurate phylogeny-based protein function prediction

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

    Sahraeian, Sayed M.; Luo, Kevin R.; Brenner, Steven E.

    We are awash in proteins discovered through high-throughput sequencing projects. As only a minuscule fraction of these have been experimentally characterized, computational methods are widely used for automated annotation. Here, we introduce a user-friendly web interface for accurate protein function prediction using the SIFTER algorithm. SIFTER is a state-of-the-art sequence-based gene molecular function prediction algorithm that uses a statistical model of function evolution to incorporate annotations throughout the phylogenetic tree. Due to the resources needed by the SIFTER algorithm, running SIFTER locally is not trivial for most users, especially for large-scale problems. The SIFTER web server thus provides access tomore » precomputed predictions on 16 863 537 proteins from 232 403 species. Users can explore SIFTER predictions with queries for proteins, species, functions, and homologs of sequences not in the precomputed prediction set. Lastly, the SIFTER web server is accessible at http://sifter.berkeley.edu/ and the source code can be downloaded.« less

  4. SIFTER search: a web server for accurate phylogeny-based protein function prediction

    DOE PAGES

    Sahraeian, Sayed M.; Luo, Kevin R.; Brenner, Steven E.

    2015-05-15

    We are awash in proteins discovered through high-throughput sequencing projects. As only a minuscule fraction of these have been experimentally characterized, computational methods are widely used for automated annotation. Here, we introduce a user-friendly web interface for accurate protein function prediction using the SIFTER algorithm. SIFTER is a state-of-the-art sequence-based gene molecular function prediction algorithm that uses a statistical model of function evolution to incorporate annotations throughout the phylogenetic tree. Due to the resources needed by the SIFTER algorithm, running SIFTER locally is not trivial for most users, especially for large-scale problems. The SIFTER web server thus provides access tomore » precomputed predictions on 16 863 537 proteins from 232 403 species. Users can explore SIFTER predictions with queries for proteins, species, functions, and homologs of sequences not in the precomputed prediction set. Lastly, the SIFTER web server is accessible at http://sifter.berkeley.edu/ and the source code can be downloaded.« less

  5. MICCA: a complete and accurate software for taxonomic profiling of metagenomic data

    PubMed Central

    Albanese, Davide; Fontana, Paolo; De Filippo, Carlotta; Cavalieri, Duccio; Donati, Claudio

    2015-01-01

    The introduction of high throughput sequencing technologies has triggered an increase of the number of studies in which the microbiota of environmental and human samples is characterized through the sequencing of selected marker genes. While experimental protocols have undergone a process of standardization that makes them accessible to a large community of scientist, standard and robust data analysis pipelines are still lacking. Here we introduce MICCA, a software pipeline for the processing of amplicon metagenomic datasets that efficiently combines quality filtering, clustering of Operational Taxonomic Units (OTUs), taxonomy assignment and phylogenetic tree inference. MICCA provides accurate results reaching a good compromise among modularity and usability. Moreover, we introduce a de-novo clustering algorithm specifically designed for the inference of Operational Taxonomic Units (OTUs). Tests on real and synthetic datasets shows that thanks to the optimized reads filtering process and to the new clustering algorithm, MICCA provides estimates of the number of OTUs and of other common ecological indices that are more accurate and robust than currently available pipelines. Analysis of public metagenomic datasets shows that the higher consistency of results improves our understanding of the structure of environmental and human associated microbial communities. MICCA is an open source project. PMID:25988396

  6. Classification of HCV and HIV-1 Sequences with the Branching Index

    PubMed Central

    Hraber, Peter; Kuiken, Carla; Waugh, Mark; Geer, Shaun; Bruno, William J.; Leitner, Thomas

    2009-01-01

    SUMMARY Classification of viral sequences should be fast, objective, accurate, and reproducible. Most methods that classify sequences use either pairwise distances or phylogenetic relations, but cannot discern when a sequence is unclassifiable. The branching index (BI) combines distance and phylogeny methods to compute a ratio that quantifies how closely a query sequence clusters with a subtype clade. In the hypothesis-testing framework of statistical inference, the BI is compared with a threshold to test whether sufficient evidence exists for the query sequence to be classified among known sequences. If above the threshold, the null hypothesis of no support for the subtype relation is rejected and the sequence is taken as belonging to the subtype clade with which it clusters on the tree. This study evaluates statistical properties of the branching index for subtype classification in HCV and HIV-1. Pairs of BI values with known positive and negative test results were computed from 10,000 random fragments of reference alignments. Sampled fragments were of sufficient length to contain phylogenetic signal that groups reference sequences together properly into subtype clades. For HCV, a threshold BI of 0.71 yields 95.1% agreement with reference subtypes, with equal false positive and false negative rates. For HIV-1, a threshold of 0.66 yields 93.5% agreement. Higher thresholds can be used where lower false positive rates are required. In synthetic recombinants, regions without breakpoints are recognized accurately; regions with breakpoints do not uniquely represent any known subtype. Web-based services for viral subtype classification with the branching index are available online. PMID:18753218

  7. Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

    PubMed Central

    Blomquist, Thomas M.; Crawford, Erin L.; Lovett, Jennie L.; Yeo, Jiyoun; Stanoszek, Lauren M.; Levin, Albert; Li, Jia; Lu, Mei; Shi, Leming; Muldrew, Kenneth; Willey, James C.

    2013-01-01

    Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1

  8. Rapid Polymer Sequencer

    NASA Technical Reports Server (NTRS)

    Stolc, Viktor (Inventor); Brock, Mathew W. (Inventor)

    2011-01-01

    Method and system for rapid and accurate determination of each of a sequence of unknown polymer components, such as nucleic acid components. A self-assembling monolayer of a selected substance is optionally provided on an interior surface of a pipette tip, and the interior surface is immersed in a selected liquid. A selected electrical field is impressed in a longitudinal or transverse direction at the tip, a polymer sequence is passed through the tip, and a change in an electrical current signal is measured as each polymer component passes through the tip. Each measured change in electrical current signals is compared with a database of reference signals, with each reference signal identified with a polymer component, to identify the unknown polymer component. The tip preferably has a pore inner diameter of no more than about 40 nm and is prepared by heating and pulling a very small section of a glass tubing.

  9. Use of low-coverage, large-insert, short-read data for rapid and accurate generation of enhanced-quality draft Pseudomonas genome sequences.

    PubMed

    O'Brien, Heath E; Gong, Yunchen; Fung, Pauline; Wang, Pauline W; Guttman, David S

    2011-01-01

    Next-generation genomic technology has both greatly accelerated the pace of genome research as well as increased our reliance on draft genome sequences. While groups such as the Genomics Standards Consortium have made strong efforts to promote genome standards there is a still a general lack of uniformity among published draft genomes, leading to challenges for downstream comparative analyses. This lack of uniformity is a particular problem when using standard draft genomes that frequently have large numbers of low-quality sequencing tracts. Here we present a proposal for an "enhanced-quality draft" genome that identifies at least 95% of the coding sequences, thereby effectively providing a full accounting of the genic component of the genome. Enhanced-quality draft genomes are easily attainable through a combination of small- and large-insert next-generation, paired-end sequencing. We illustrate the generation of an enhanced-quality draft genome by re-sequencing the plant pathogenic bacterium Pseudomonas syringae pv. phaseolicola 1448A (Pph 1448A), which has a published, closed genome sequence of 5.93 Mbp. We use a combination of Illumina paired-end and mate-pair sequencing, and surprisingly find that de novo assemblies with 100x paired-end coverage and mate-pair sequencing with as low as low as 2-5x coverage are substantially better than assemblies based on higher coverage. The rapid and low-cost generation of large numbers of enhanced-quality draft genome sequences will be of particular value for microbial diagnostics and biosecurity, which rely on precise discrimination of potentially dangerous clones from closely related benign strains.

  10. Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis.

    PubMed

    Jakupciak, John P; Wells, Jeffrey M; Karalus, Richard J; Pawlowski, David R; Lin, Jeffrey S; Feldman, Andrew B

    2013-01-01

    Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.

  11. Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis

    PubMed Central

    Jakupciak, John P.; Wells, Jeffrey M.; Karalus, Richard J.; Pawlowski, David R.; Lin, Jeffrey S.; Feldman, Andrew B.

    2013-01-01

    Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations. PMID:24455204

  12. DNA demethylation activates genes in seed maternal integument development in rice (Oryza sativa L.).

    PubMed

    Wang, Yifeng; Lin, Haiyan; Tong, Xiaohong; Hou, Yuxuan; Chang, Yuxiao; Zhang, Jian

    2017-11-01

    DNA methylation is an important epigenetic modification that regulates various plant developmental processes. Rice seed integument determines the seed size. However, the role of DNA methylation in its development remains largely unknown. Here, we report the first dynamic DNA methylomic profiling of rice maternal integument before and after pollination by using a whole-genome bisulfite deep sequencing approach. Analysis of DNA methylation patterns identified 4238 differentially methylated regions underpin 4112 differentially methylated genes, including GW2, DEP1, RGB1 and numerous other regulators participated in maternal integument development. Bisulfite sanger sequencing and qRT-PCR of six differentially methylated genes revealed extensive occurrence of DNA hypomethylation triggered by double fertilization at IAP compared with IBP, suggesting that DNA demethylation might be a key mechanism to activate numerous maternal controlling genes. These results presented here not only greatly expanded the rice methylome dataset, but also shed novel insight into the regulatory roles of DNA methylation in rice seed maternal integument development. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

    PubMed Central

    Sun, Kun; Jiang, Peiyong; Chan, K. C. Allen; Wong, John; Cheng, Yvonne K. Y.; Liang, Raymond H. S.; Chan, Wai-kong; Ma, Edmond S. K.; Chan, Stephen L.; Cheng, Suk Hang; Chan, Rebecca W. Y.; Tong, Yu K.; Ng, Simon S. M.; Wong, Raymond S. M.; Hui, David S. C.; Leung, Tse Ngong; Leung, Tak Y.; Lai, Paul B. S.; Chiu, Rossa W. K.; Lo, Yuk Ming Dennis

    2015-01-01

    Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma. PMID:26392541

  14. Protein 3D Structure Computed from Evolutionary Sequence Variation

    PubMed Central

    Sheridan, Robert; Hopf, Thomas A.; Pagnani, Andrea; Zecchina, Riccardo; Sander, Chris

    2011-01-01

    The evolutionary trajectory of a protein through sequence space is constrained by its function. Collections of sequence homologs record the outcomes of millions of evolutionary experiments in which the protein evolves according to these constraints. Deciphering the evolutionary record held in these sequences and exploiting it for predictive and engineering purposes presents a formidable challenge. The potential benefit of solving this challenge is amplified by the advent of inexpensive high-throughput genomic sequencing. In this paper we ask whether we can infer evolutionary constraints from a set of sequence homologs of a protein. The challenge is to distinguish true co-evolution couplings from the noisy set of observed correlations. We address this challenge using a maximum entropy model of the protein sequence, constrained by the statistics of the multiple sequence alignment, to infer residue pair couplings. Surprisingly, we find that the strength of these inferred couplings is an excellent predictor of residue-residue proximity in folded structures. Indeed, the top-scoring residue couplings are sufficiently accurate and well-distributed to define the 3D protein fold with remarkable accuracy. We quantify this observation by computing, from sequence alone, all-atom 3D structures of fifteen test proteins from different fold classes, ranging in size from 50 to 260 residues., including a G-protein coupled receptor. These blinded inferences are de novo, i.e., they do not use homology modeling or sequence-similar fragments from known structures. The co-evolution signals provide sufficient information to determine accurate 3D protein structure to 2.7–4.8 Å Cα-RMSD error relative to the observed structure, over at least two-thirds of the protein (method called EVfold, details at http://EVfold.org). This discovery provides insight into essential interactions constraining protein evolution and will facilitate a comprehensive survey of the universe of protein

  15. Preferential access to genetic information from endogenous hominin ancient DNA and accurate quantitative SNP-typing via SPEX

    PubMed Central

    Brotherton, Paul; Sanchez, Juan J.; Cooper, Alan; Endicott, Phillip

    2010-01-01

    The analysis of targeted genetic loci from ancient, forensic and clinical samples is usually built upon polymerase chain reaction (PCR)-generated sequence data. However, many studies have shown that PCR amplification from poor-quality DNA templates can create sequence artefacts at significant levels. With hominin (human and other hominid) samples, the pervasive presence of highly PCR-amplifiable human DNA contaminants in the vast majority of samples can lead to the creation of recombinant hybrids and other non-authentic artefacts. The resulting PCR-generated sequences can then be difficult, if not impossible, to authenticate. In contrast, single primer extension (SPEX)-based approaches can genotype single nucleotide polymorphisms from ancient fragments of DNA as accurately as modern DNA. A single SPEX-type assay can amplify just one of the duplex DNA strands at target loci and generate a multi-fold depth-of-coverage, with non-authentic recombinant hybrids reduced to undetectable levels. Crucially, SPEX-type approaches can preferentially access genetic information from damaged and degraded endogenous ancient DNA templates over modern human DNA contaminants. The development of SPEX-type assays offers the potential for highly accurate, quantitative genotyping from ancient hominin samples. PMID:19864251

  16. Calibrating genomic and allelic coverage bias in single-cell sequencing.

    PubMed

    Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher

    2015-04-16

    Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.

  17. Calibrating genomic and allelic coverage bias in single-cell sequencing

    PubMed Central

    Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher

    2016-01-01

    Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913

  18. DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing.

    PubMed

    Castle, John C; Biery, Matthew; Bouzek, Heather; Xie, Tao; Chen, Ronghua; Misura, Kira; Jackson, Stuart; Armour, Christopher D; Johnson, Jason M; Rohl, Carol A; Raymond, Christopher K

    2010-04-16

    DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads.

  19. High-Quality Genome Sequence of the Highly Resistant Bacterium Staphylococcus haemolyticus, Isolated from a Neonatal Bloodstream Infection.

    PubMed

    Hosseinkhani, Farideh; Emaneini, Mohammad; van Leeuwen, Willem

    2017-07-20

    Using Illumina HiSeq and PacBio technologies, we sequenced the genome of the multidrug-resistant bacterium Staphylococcus haemolyticus , originating from a bloodstream infection in a neonate. The sequence data can be used as an accurate reference sequence. Copyright © 2017 Hosseinkhani et al.

  20. 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

  1. A photogrammetric technique for generation of an accurate multispectral optical flow dataset

    NASA Astrophysics Data System (ADS)

    Kniaz, V. V.

    2017-06-01

    A presence of an accurate dataset is the key requirement for a successful development of an optical flow estimation algorithm. A large number of freely available optical flow datasets were developed in recent years and gave rise for many powerful algorithms. However most of the datasets include only images captured in the visible spectrum. This paper is focused on the creation of a multispectral optical flow dataset with an accurate ground truth. The generation of an accurate ground truth optical flow is a rather complex problem, as no device for error-free optical flow measurement was developed to date. Existing methods for ground truth optical flow estimation are based on hidden textures, 3D modelling or laser scanning. Such techniques are either work only with a synthetic optical flow or provide a sparse ground truth optical flow. In this paper a new photogrammetric method for generation of an accurate ground truth optical flow is proposed. The method combines the benefits of the accuracy and density of a synthetic optical flow datasets with the flexibility of laser scanning based techniques. A multispectral dataset including various image sequences was generated using the developed method. The dataset is freely available on the accompanying web site.

  2. Correcting for sequencing error in maximum likelihood phylogeny inference.

    PubMed

    Kuhner, Mary K; McGill, James

    2014-11-04

    Accurate phylogenies are critical to taxonomy as well as studies of speciation processes and other evolutionary patterns. Accurate branch lengths in phylogenies are critical for dating and rate measurements. Such accuracy may be jeopardized by unacknowledged sequencing error. We use simulated data to test a correction for DNA sequencing error in maximum likelihood phylogeny inference. Over a wide range of data polymorphism and true error rate, we found that correcting for sequencing error improves recovery of the branch lengths, even if the assumed error rate is up to twice the true error rate. Low error rates have little effect on recovery of the topology. When error is high, correction improves topological inference; however, when error is extremely high, using an assumed error rate greater than the true error rate leads to poor recovery of both topology and branch lengths. The error correction approach tested here was proposed in 2004 but has not been widely used, perhaps because researchers do not want to commit to an estimate of the error rate. This study shows that correction with an approximate error rate is generally preferable to ignoring the issue. Copyright © 2014 Kuhner and McGill.

  3. How Messenger RNA and Nascent Chain Sequences Regulate Translation Elongation.

    PubMed

    Choi, Junhong; Grosely, Rosslyn; Prabhakar, Arjun; Lapointe, Christopher P; Wang, Jinfan; Puglisi, Joseph D

    2018-06-20

    Translation elongation is a highly coordinated, multistep, multifactor process that ensures accurate and efficient addition of amino acids to a growing nascent-peptide chain encoded in the sequence of translated messenger RNA (mRNA). Although translation elongation is heavily regulated by external factors, there is clear evidence that mRNA and nascent-peptide sequences control elongation dynamics, determining both the sequence and structure of synthesized proteins. Advances in methods have driven experiments that revealed the basic mechanisms of elongation as well as the mechanisms of regulation by mRNA and nascent-peptide sequences. In this review, we highlight how mRNA and nascent-peptide elements manipulate the translation machinery to alter the dynamics and pathway of elongation.

  4. Protein Sequencing with Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Ziady, Assem G.; Kinter, Michael

    The recent introduction of electrospray ionization techniques that are suitable for peptides and whole proteins has allowed for the design of mass spectrometric protocols that provide accurate sequence information for proteins. The advantages gained by these approaches over traditional Edman Degradation sequencing include faster analysis and femtomole, sometimes attomole, sensitivity. The ability to efficiently identify proteins has allowed investigators to conduct studies on their differential expression or modification in response to various treatments or disease states. In this chapter, we discuss the use of electrospray tandem mass spectrometry, a technique whereby protein-derived peptides are subjected to fragmentation in the gas phase, revealing sequence information for the protein. This powerful technique has been instrumental for the study of proteins and markers associated with various disorders, including heart disease, cancer, and cystic fibrosis. We use the study of protein expression in cystic fibrosis as an example.

  5. Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium.

    PubMed

    Pelch, Katherine E; Tokar, Erik J; Merrick, B Alex; Waalkes, Michael P

    2015-08-01

    Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. Published by Elsevier Inc.

  6. Iterative refinement of structure-based sequence alignments by Seed Extension

    PubMed Central

    Kim, Changhoon; Tai, Chin-Hsien; Lee, Byungkook

    2009-01-01

    Background Accurate sequence alignment is required in many bioinformatics applications but, when sequence similarity is low, it is difficult to obtain accurate alignments based on sequence similarity alone. The accuracy improves when the structures are available, but current structure-based sequence alignment procedures still mis-align substantial numbers of residues. In order to correct such errors, we previously explored the possibility of replacing the residue-based dynamic programming algorithm in structure alignment procedures with the Seed Extension algorithm, which does not use a gap penalty. Here, we describe a new procedure called RSE (Refinement with Seed Extension) that iteratively refines a structure-based sequence alignment. Results RSE uses SE (Seed Extension) in its core, which is an algorithm that we reported recently for obtaining a sequence alignment from two superimposed structures. The RSE procedure was evaluated by comparing the correctly aligned fractions of residues before and after the refinement of the structure-based sequence alignments produced by popular programs. CE, DaliLite, FAST, LOCK2, MATRAS, MATT, TM-align, SHEBA and VAST were included in this analysis and the NCBI's CDD root node set was used as the reference alignments. RSE improved the average accuracy of sequence alignments for all programs tested when no shift error was allowed. The amount of improvement varied depending on the program. The average improvements were small for DaliLite and MATRAS but about 5% for CE and VAST. More substantial improvements have been seen in many individual cases. The additional computation times required for the refinements were negligible compared to the times taken by the structure alignment programs. Conclusion RSE is a computationally inexpensive way of improving the accuracy of a structure-based sequence alignment. It can be used as a standalone procedure following a regular structure-based sequence alignment or to replace the traditional

  7. Impact of sequencing depth and read length on single cell RNA sequencing data of T cells.

    PubMed

    Rizzetto, Simone; Eltahla, Auda A; Lin, Peijie; Bull, Rowena; Lloyd, Andrew R; Ho, Joshua W K; Venturi, Vanessa; Luciani, Fabio

    2017-10-06

    Single cell RNA sequencing (scRNA-seq) provides great potential in measuring the gene expression profiles of heterogeneous cell populations. In immunology, scRNA-seq allowed the characterisation of transcript sequence diversity of functionally relevant T cell subsets, and the identification of the full length T cell receptor (TCRαβ), which defines the specificity against cognate antigens. Several factors, e.g. RNA library capture, cell quality, and sequencing output affect the quality of scRNA-seq data. We studied the effects of read length and sequencing depth on the quality of gene expression profiles, cell type identification, and TCRαβ reconstruction, utilising 1,305 single cells from 8 publically available scRNA-seq datasets, and simulation-based analyses. Gene expression was characterised by an increased number of unique genes identified with short read lengths (<50 bp), but these featured higher technical variability compared to profiles from longer reads. Successful TCRαβ reconstruction was achieved for 6 datasets (81% - 100%) with at least 0.25 millions (PE) reads of length >50 bp, while it failed for datasets with <30 bp reads. Sufficient read length and sequencing depth can control technical noise to enable accurate identification of TCRαβ and gene expression profiles from scRNA-seq data of T cells.

  8. FastaValidator: an open-source Java library to parse and validate FASTA formatted sequences.

    PubMed

    Waldmann, Jost; Gerken, Jan; Hankeln, Wolfgang; Schweer, Timmy; Glöckner, Frank Oliver

    2014-06-14

    Advances in sequencing technologies challenge the efficient importing and validation of FASTA formatted sequence data which is still a prerequisite for most bioinformatic tools and pipelines. Comparative analysis of commonly used Bio*-frameworks (BioPerl, BioJava and Biopython) shows that their scalability and accuracy is hampered. FastaValidator represents a platform-independent, standardized, light-weight software library written in the Java programming language. It targets computer scientists and bioinformaticians writing software which needs to parse quickly and accurately large amounts of sequence data. For end-users FastaValidator includes an interactive out-of-the-box validation of FASTA formatted files, as well as a non-interactive mode designed for high-throughput validation in software pipelines. The accuracy and performance of the FastaValidator library qualifies it for large data sets such as those commonly produced by massive parallel (NGS) technologies. It offers scientists a fast, accurate and standardized method for parsing and validating FASTA formatted sequence data.

  9. Asymmetric single-strand polymorphism: an accurate and cost-effective method to amplify and sequence allelic variants

    USDA-ARS?s Scientific Manuscript database

    We needed to obtain an alternative to conventional cloning to generate high-quality DNA sequences from a variety of nuclear orthologs for phylogenetic studies in potato, to save time and money and to avoid problems typically encountered in cloning. We tested a variety of SSCP protocols to include pu...

  10. Enduring epigenetic landmarks define the cancer microenvironment

    PubMed Central

    Pidsley, Ruth; Lawrence, Mitchell G.; Zotenko, Elena; Niranjan, Birunthi; Statham, Aaron; Song, Jenny; Chabanon, Roman M.; Qu, Wenjia; Wang, Hong; Richards, Michelle; Nair, Shalima S.; Armstrong, Nicola J.; Nim, Hieu T.; Papargiris, Melissa; Balanathan, Preetika; French, Hugh; Peters, Timothy; Norden, Sam; Ryan, Andrew; Pedersen, John; Kench, James; Daly, Roger J.; Horvath, Lisa G.; Stricker, Phillip; Frydenberg, Mark; Taylor, Renea A.; Stirzaker, Clare; Risbridger, Gail P.; Clark, Susan J.

    2018-01-01

    The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples. PMID:29650553

  11. Bacterial identification and subtyping using DNA microarray and DNA sequencing.

    PubMed

    Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

    2012-01-01

    The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

  12. Predicting disulfide connectivity from protein sequence using multiple sequence feature vectors and secondary structure.

    PubMed

    Song, Jiangning; Yuan, Zheng; Tan, Hao; Huber, Thomas; Burrage, Kevin

    2007-12-01

    Disulfide bonds are primary covalent crosslinks between two cysteine residues in proteins that play critical roles in stabilizing the protein structures and are commonly found in extracy-toplasmatic or secreted proteins. In protein folding prediction, the localization of disulfide bonds can greatly reduce the search in conformational space. Therefore, there is a great need to develop computational methods capable of accurately predicting disulfide connectivity patterns in proteins that could have potentially important applications. We have developed a novel method to predict disulfide connectivity patterns from protein primary sequence, using a support vector regression (SVR) approach based on multiple sequence feature vectors and predicted secondary structure by the PSIPRED program. The results indicate that our method could achieve a prediction accuracy of 74.4% and 77.9%, respectively, when averaged on proteins with two to five disulfide bridges using 4-fold cross-validation, measured on the protein and cysteine pair on a well-defined non-homologous dataset. We assessed the effects of different sequence encoding schemes on the prediction performance of disulfide connectivity. It has been shown that the sequence encoding scheme based on multiple sequence feature vectors coupled with predicted secondary structure can significantly improve the prediction accuracy, thus enabling our method to outperform most of other currently available predictors. Our work provides a complementary approach to the current algorithms that should be useful in computationally assigning disulfide connectivity patterns and helps in the annotation of protein sequences generated by large-scale whole-genome projects. The prediction web server and Supplementary Material are accessible at http://foo.maths.uq.edu.au/~huber/disulfide

  13. Fast imputation using medium or low-coverage sequence data

    USDA-ARS?s Scientific Manuscript database

    Accurate genotype imputation can greatly reduce costs and increase benefits by combining whole-genome sequence data of varying read depth and microarray genotypes of varying densities. For large populations, an efficient strategy chooses the two haplotypes most likely to form each genotype and updat...

  14. Multilocus sequence analysis and rpoB sequencing of Mycobacterium abscessus (sensu lato) strains.

    PubMed

    Macheras, Edouard; Roux, Anne-Laure; Bastian, Sylvaine; Leão, Sylvia Cardoso; Palaci, Moises; Sivadon-Tardy, Valérie; Gutierrez, Cristina; Richter, Elvira; Rüsch-Gerdes, Sabine; Pfyffer, Gaby; Bodmer, Thomas; Cambau, Emmanuelle; Gaillard, Jean-Louis; Heym, Beate

    2011-02-01

    clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

  15. Multilocus Sequence Analysis and rpoB Sequencing of Mycobacterium abscessus (Sensu Lato) Strains▿

    PubMed Central

    Macheras, Edouard; Roux, Anne-Laure; Bastian, Sylvaine; Leão, Sylvia Cardoso; Palaci, Moises; Sivadon-Tardy, Valérie; Gutierrez, Cristina; Richter, Elvira; Rüsch-Gerdes, Sabine; Pfyffer, Gaby; Bodmer, Thomas; Cambau, Emmanuelle; Gaillard, Jean-Louis; Heym, Beate

    2011-01-01

    of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies. PMID:21106786

  16. DNA copy number, including telomeres and mitochondria, assayed using next-generation sequencing

    PubMed Central

    2010-01-01

    Background DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. Results We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. Conclusion The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads. PMID:20398377

  17. De novo assembly of human genomes with massively parallel short read sequencing.

    PubMed

    Li, Ruiqiang; Zhu, Hongmei; Ruan, Jue; Qian, Wubin; Fang, Xiaodong; Shi, Zhongbin; Li, Yingrui; Li, Shengting; Shan, Gao; Kristiansen, Karsten; Li, Songgang; Yang, Huanming; Wang, Jian; Wang, Jun

    2010-02-01

    Next-generation massively parallel DNA sequencing technologies provide ultrahigh throughput at a substantially lower unit data cost; however, the data are very short read length sequences, making de novo assembly extremely challenging. Here, we describe a novel method for de novo assembly of large genomes from short read sequences. We successfully assembled both the Asian and African human genome sequences, achieving an N50 contig size of 7.4 and 5.9 kilobases (kb) and scaffold of 446.3 and 61.9 kb, respectively. The development of this de novo short read assembly method creates new opportunities for building reference sequences and carrying out accurate analyses of unexplored genomes in a cost-effective way.

  18. Enhanced sequencing coverage with digital droplet multiple displacement amplification

    PubMed Central

    Sidore, Angus M.; Lan, Freeman; Lim, Shaun W.; Abate, Adam R.

    2016-01-01

    Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing. PMID:26704978

  19. Rapid Polymer Sequencer

    NASA Technical Reports Server (NTRS)

    Stolc, Viktor (Inventor); Brock, Matthew W (Inventor)

    2013-01-01

    Method and system for rapid and accurate determination of each of a sequence of unknown polymer components, such as nucleic acid components. A self-assembling monolayer of a selected substance is optionally provided on an interior surface of a pipette tip, and the interior surface is immersed in a selected liquid. A selected electrical field is impressed in a longitudinal direction, or in a transverse direction, in the tip region, a polymer sequence is passed through the tip region, and a change in an electrical current signal is measured as each polymer component passes through the tip region. Each of the measured changes in electrical current signals is compared with a database of reference electrical change signals, with each reference signal corresponding to an identified polymer component, to identify the unknown polymer component with a reference polymer component. The nanopore preferably has a pore inner diameter of no more than about 40 nm and is prepared by heating and pulling a very small section of a glass tubing.

  20. Sequencing our way towards understanding global eukaryotic biodiversity

    PubMed Central

    Bik, Holly M.; Porazinska, Dorota L.; Creer, Simon; Caporaso, J. Gregory; Knight, Rob; Thomas, W. Kelley

    2011-01-01

    Microscopic eukaryotes are abundant, diverse, and fill critical ecological roles across every ecosystem on earth, yet there is a well-recognized gap in our understanding of their global biodiversity. Fundamental advances in DNA sequencing and bioinformatics now allow accurate en masse biodiversity assessments of microscopic eukaryotes from environmental samples. Despite a promising outlook, the field of eukaryotic marker gene surveys faces significant challenges: how to generate data that is most useful to the community, especially in the face of evolving sequencing technology and bioinformatics pipelines, and how to incorporate an expanding number of target genes. PMID:22244672

  1. BLESS 2: accurate, memory-efficient and fast error correction method.

    PubMed

    Heo, Yun; Ramachandran, Anand; Hwu, Wen-Mei; Ma, Jian; Chen, Deming

    2016-08-01

    The most important features of error correction tools for sequencing data are accuracy, memory efficiency and fast runtime. The previous version of BLESS was highly memory-efficient and accurate, but it was too slow to handle reads from large genomes. We have developed a new version of BLESS to improve runtime and accuracy while maintaining a small memory usage. The new version, called BLESS 2, has an error correction algorithm that is more accurate than BLESS, and the algorithm has been parallelized using hybrid MPI and OpenMP programming. BLESS 2 was compared with five top-performing tools, and it was found to be the fastest when it was executed on two computing nodes using MPI, with each node containing twelve cores. Also, BLESS 2 showed at least 11% higher gain while retaining the memory efficiency of the previous version for large genomes. Freely available at https://sourceforge.net/projects/bless-ec dchen@illinois.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.

  2. On site DNA barcoding by nanopore sequencing

    PubMed Central

    Menegon, Michele; Cantaloni, Chiara; Rodriguez-Prieto, Ana; Centomo, Cesare; Abdelfattah, Ahmed; Rossato, Marzia; Bernardi, Massimo; Xumerle, Luciano; Loader, Simon; Delledonne, Massimo

    2017-01-01

    Biodiversity research is becoming increasingly dependent on genomics, which allows the unprecedented digitization and understanding of the planet’s biological heritage. The use of genetic markers i.e. DNA barcoding, has proved to be a powerful tool in species identification. However, full exploitation of this approach is hampered by the high sequencing costs and the absence of equipped facilities in biodiversity-rich countries. In the present work, we developed a portable sequencing laboratory based on the portable DNA sequencer from Oxford Nanopore Technologies, the MinION. Complementary laboratory equipment and reagents were selected to be used in remote and tough environmental conditions. The performance of the MinION sequencer and the portable laboratory was tested for DNA barcoding in a mimicking tropical environment, as well as in a remote rainforest of Tanzania lacking electricity. Despite the relatively high sequencing error-rate of the MinION, the development of a suitable pipeline for data analysis allowed the accurate identification of different species of vertebrates including amphibians, reptiles and mammals. In situ sequencing of a wild frog allowed us to rapidly identify the species captured, thus confirming that effective DNA barcoding in the field is possible. These results open new perspectives for real-time-on-site DNA sequencing thus potentially increasing opportunities for the understanding of biodiversity in areas lacking conventional laboratory facilities. PMID:28977016

  3. A multiplex microplatform for the detection of multiple DNA methylation events using gold-DNA affinity.

    PubMed

    Sina, Abu Ali Ibn; Foster, Matthew Thomas; Korbie, Darren; Carrascosa, Laura G; Shiddiky, Muhammad J A; Gao, Jing; Dey, Shuvashis; Trau, Matt

    2017-10-07

    We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg μl -1 of DNA with no sequencing requirement.

  4. Re-Ranking Sequencing Variants in the Post-GWAS Era for Accurate Causal Variant Identification

    PubMed Central

    Faye, Laura L.; Machiela, Mitchell J.; Kraft, Peter; Bull, Shelley B.; Sun, Lei

    2013-01-01

    Next generation sequencing has dramatically increased our ability to localize disease-causing variants by providing base-pair level information at costs increasingly feasible for the large sample sizes required to detect complex-trait associations. Yet, identification of causal variants within an established region of association remains a challenge. Counter-intuitively, certain factors that increase power to detect an associated region can decrease power to localize the causal variant. First, combining GWAS with imputation or low coverage sequencing to achieve the large sample sizes required for high power can have the unintended effect of producing differential genotyping error among SNPs. This tends to bias the relative evidence for association toward better genotyped SNPs. Second, re-use of GWAS data for fine-mapping exploits previous findings to ensure genome-wide significance in GWAS-associated regions. However, using GWAS findings to inform fine-mapping analysis can bias evidence away from the causal SNP toward the tag SNP and SNPs in high LD with the tag. Together these factors can reduce power to localize the causal SNP by more than half. Other strategies commonly employed to increase power to detect association, namely increasing sample size and using higher density genotyping arrays, can, in certain common scenarios, actually exacerbate these effects and further decrease power to localize causal variants. We develop a re-ranking procedure that accounts for these adverse effects and substantially improves the accuracy of causal SNP identification, often doubling the probability that the causal SNP is top-ranked. Application to the NCI BPC3 aggressive prostate cancer GWAS with imputation meta-analysis identified a new top SNP at 2 of 3 associated loci and several additional possible causal SNPs at these loci that may have otherwise been overlooked. This method is simple to implement using R scripts provided on the author's website. PMID:23950724

  5. Recommendations for Accurate Resolution of Gene and Isoform Allele-Specific Expression in RNA-Seq Data

    PubMed Central

    Wood, David L. A.; Nones, Katia; Steptoe, Anita; Christ, Angelika; Harliwong, Ivon; Newell, Felicity; Bruxner, Timothy J. C.; Miller, David; Cloonan, Nicole; Grimmond, Sean M.

    2015-01-01

    Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual’s phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci. PMID:25965996

  6. Fast imputation using medium- or low-coverage sequence data

    USDA-ARS?s Scientific Manuscript database

    Direct imputation from raw sequence reads can be more accurate than calling genotypes first and then imputing, especially if read depth is low or error rates high, but different imputation strategies are required than those used for data from genotyping chips. A fast algorithm to impute from lower t...

  7. Robustness of Massively Parallel Sequencing Platforms

    PubMed Central

    Kavak, Pınar; Yüksel, Bayram; Aksu, Soner; Kulekci, M. Oguzhan; Güngör, Tunga; Hach, Faraz; Şahinalp, S. Cenk; Alkan, Can; Sağıroğlu, Mahmut Şamil

    2015-01-01

    The improvements in high throughput sequencing technologies (HTS) made clinical sequencing projects such as ClinSeq and Genomics England feasible. Although there are significant improvements in accuracy and reproducibility of HTS based analyses, the usability of these types of data for diagnostic and prognostic applications necessitates a near perfect data generation. To assess the usability of a widely used HTS platform for accurate and reproducible clinical applications in terms of robustness, we generated whole genome shotgun (WGS) sequence data from the genomes of two human individuals in two different genome sequencing centers. After analyzing the data to characterize SNPs and indels using the same tools (BWA, SAMtools, and GATK), we observed significant number of discrepancies in the call sets. As expected, the most of the disagreements between the call sets were found within genomic regions containing common repeats and segmental duplications, albeit only a small fraction of the discordant variants were within the exons and other functionally relevant regions such as promoters. We conclude that although HTS platforms are sufficiently powerful for providing data for first-pass clinical tests, the variant predictions still need to be confirmed using orthogonal methods before using in clinical applications. PMID:26382624

  8. Integrative structural annotation of de novo RNA-Seq provides an accurate reference gene set of the enormous genome of the onion (Allium cepa L.)

    PubMed Central

    Kim, Seungill; Kim, Myung-Shin; Kim, Yong-Min; Yeom, Seon-In; Cheong, Kyeongchae; Kim, Ki-Tae; Jeon, Jongbum; Kim, Sunggil; Kim, Do-Sun; Sohn, Seong-Han; Lee, Yong-Hwan; Choi, Doil

    2015-01-01

    The onion (Allium cepa L.) is one of the most widely cultivated and consumed vegetable crops in the world. Although a considerable amount of onion transcriptome data has been deposited into public databases, the sequences of the protein-coding genes are not accurate enough to be used, owing to non-coding sequences intermixed with the coding sequences. We generated a high-quality, annotated onion transcriptome from de novo sequence assembly and intensive structural annotation using the integrated structural gene annotation pipeline (ISGAP), which identified 54,165 protein-coding genes among 165,179 assembled transcripts totalling 203.0 Mb by eliminating the intron sequences. ISGAP performed reliable annotation, recognizing accurate gene structures based on reference proteins, and ab initio gene models of the assembled transcripts. Integrative functional annotation and gene-based SNP analysis revealed a whole biological repertoire of genes and transcriptomic variation in the onion. The method developed in this study provides a powerful tool for the construction of reference gene sets for organisms based solely on de novo transcriptome data. Furthermore, the reference genes and their variation described here for the onion represent essential tools for molecular breeding and gene cloning in Allium spp. PMID:25362073

  9. Iterative Correction of Reference Nucleotides (iCORN) using second generation sequencing technology.

    PubMed

    Otto, Thomas D; Sanders, Mandy; Berriman, Matthew; Newbold, Chris

    2010-07-15

    The accuracy of reference genomes is important for downstream analysis but a low error rate requires expensive manual interrogation of the sequence. Here, we describe a novel algorithm (Iterative Correction of Reference Nucleotides) that iteratively aligns deep coverage of short sequencing reads to correct errors in reference genome sequences and evaluate their accuracy. Using Plasmodium falciparum (81% A + T content) as an extreme example, we show that the algorithm is highly accurate and corrects over 2000 errors in the reference sequence. We give examples of its application to numerous other eukaryotic and prokaryotic genomes and suggest additional applications. The software is available at http://icorn.sourceforge.net

  10. MeCorS: Metagenome-enabled error correction of single cell sequencing reads

    DOE PAGES

    Bremges, Andreas; Singer, Esther; Woyke, Tanja; ...

    2016-03-15

    Here we present a new tool, MeCorS, to correct chimeric reads and sequencing errors in Illumina data generated from single amplified genomes (SAGs). It uses sequence information derived from accompanying metagenome sequencing to accurately correct errors in SAG reads, even from ultra-low coverage regions. In evaluations on real data, we show that MeCorS outperforms BayesHammer, the most widely used state-of-the-art approach. MeCorS performs particularly well in correcting chimeric reads, which greatly improves both accuracy and contiguity of de novo SAG assemblies.

  11. A segmentation method for lung nodule image sequences based on superpixels and density-based spatial clustering of applications with noise

    PubMed Central

    Zhang, Wei; Zhang, Xiaolong; Qiang, Yan; Tian, Qi; Tang, Xiaoxian

    2017-01-01

    The fast and accurate segmentation of lung nodule image sequences is the basis of subsequent processing and diagnostic analyses. However, previous research investigating nodule segmentation algorithms cannot entirely segment cavitary nodules, and the segmentation of juxta-vascular nodules is inaccurate and inefficient. To solve these problems, we propose a new method for the segmentation of lung nodule image sequences based on superpixels and density-based spatial clustering of applications with noise (DBSCAN). First, our method uses three-dimensional computed tomography image features of the average intensity projection combined with multi-scale dot enhancement for preprocessing. Hexagonal clustering and morphological optimized sequential linear iterative clustering (HMSLIC) for sequence image oversegmentation is then proposed to obtain superpixel blocks. The adaptive weight coefficient is then constructed to calculate the distance required between superpixels to achieve precise lung nodules positioning and to obtain the subsequent clustering starting block. Moreover, by fitting the distance and detecting the change in slope, an accurate clustering threshold is obtained. Thereafter, a fast DBSCAN superpixel sequence clustering algorithm, which is optimized by the strategy of only clustering the lung nodules and adaptive threshold, is then used to obtain lung nodule mask sequences. Finally, the lung nodule image sequences are obtained. The experimental results show that our method rapidly, completely and accurately segments various types of lung nodule image sequences. PMID:28880916

  12. MANGO: a new approach to multiple sequence alignment.

    PubMed

    Zhang, Zefeng; Lin, Hao; Li, Ming

    2007-01-01

    Multiple sequence alignment is a classical and challenging task for biological sequence analysis. The problem is NP-hard. The full dynamic programming takes too much time. The progressive alignment heuristics adopted by most state of the art multiple sequence alignment programs suffer from the 'once a gap, always a gap' phenomenon. Is there a radically new way to do multiple sequence alignment? This paper introduces a novel and orthogonal multiple sequence alignment method, using multiple optimized spaced seeds and new algorithms to handle these seeds efficiently. Our new algorithm processes information of all sequences as a whole, avoiding problems caused by the popular progressive approaches. Because the optimized spaced seeds are provably significantly more sensitive than the consecutive k-mers, the new approach promises to be more accurate and reliable. To validate our new approach, we have implemented MANGO: Multiple Alignment with N Gapped Oligos. Experiments were carried out on large 16S RNA benchmarks showing that MANGO compares favorably, in both accuracy and speed, against state-of-art multiple sequence alignment methods, including ClustalW 1.83, MUSCLE 3.6, MAFFT 5.861, Prob-ConsRNA 1.11, Dialign 2.2.1, DIALIGN-T 0.2.1, T-Coffee 4.85, POA 2.0 and Kalign 2.0.

  13. pyPaSWAS: Python-based multi-core CPU and GPU sequence alignment.

    PubMed

    Warris, Sven; Timal, N Roshan N; Kempenaar, Marcel; Poortinga, Arne M; van de Geest, Henri; Varbanescu, Ana L; Nap, Jan-Peter

    2018-01-01

    Our previously published CUDA-only application PaSWAS for Smith-Waterman (SW) sequence alignment of any type of sequence on NVIDIA-based GPUs is platform-specific and therefore adopted less than could be. The OpenCL language is supported more widely and allows use on a variety of hardware platforms. Moreover, there is a need to promote the adoption of parallel computing in bioinformatics by making its use and extension more simple through more and better application of high-level languages commonly used in bioinformatics, such as Python. The novel application pyPaSWAS presents the parallel SW sequence alignment code fully packed in Python. It is a generic SW implementation running on several hardware platforms with multi-core systems and/or GPUs that provides accurate sequence alignments that also can be inspected for alignment details. Additionally, pyPaSWAS support the affine gap penalty. Python libraries are used for automated system configuration, I/O and logging. This way, the Python environment will stimulate further extension and use of pyPaSWAS. pyPaSWAS presents an easy Python-based environment for accurate and retrievable parallel SW sequence alignments on GPUs and multi-core systems. The strategy of integrating Python with high-performance parallel compute languages to create a developer- and user-friendly environment should be considered for other computationally intensive bioinformatics algorithms.

  14. Sequence harmony: detecting functional specificity from alignments

    PubMed Central

    Feenstra, K. Anton; Pirovano, Walter; Krab, Klaas; Heringa, Jaap

    2007-01-01

    Multiple sequence alignments are often used for the identification of key specificity-determining residues within protein families. We present a web server implementation of the Sequence Harmony (SH) method previously introduced. SH accurately detects subfamily specific positions from a multiple alignment by scoring compositional differences between subfamilies, without imposing conservation. The SH web server allows a quick selection of subtype specific sites from a multiple alignment given a subfamily grouping. In addition, it allows the predicted sites to be directly mapped onto a protein structure and displayed. We demonstrate the use of the SH server using the family of plant mitochondrial alternative oxidases (AOX). In addition, we illustrate the usefulness of combining sequence and structural information by showing that the predicted sites are clustered into a few distinct regions in an AOX homology model. The SH web server can be accessed at www.ibi.vu.nl/programs/seqharmwww. PMID:17584793

  15. 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

  16. An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

    PubMed

    Seashols-Williams, Sarah; Green, Raquel; Wohlfahrt, Denise; Brand, Angela; Tan-Torres, Antonio Limjuco; Nogales, Francy; Brooks, J Paul; Singh, Baneshwar

    2018-05-17

    Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. cuRRBS: simple and robust evaluation of enzyme combinations for reduced representation approaches.

    PubMed

    Martin-Herranz, Daniel E; Ribeiro, António J M; Krueger, Felix; Thornton, Janet M; Reik, Wolf; Stubbs, Thomas M

    2017-11-16

    DNA methylation is an important epigenetic modification in many species that is critical for development, and implicated in ageing and many complex diseases, such as cancer. Many cost-effective genome-wide analyses of DNA modifications rely on restriction enzymes capable of digesting genomic DNA at defined sequence motifs. There are hundreds of restriction enzyme families but few are used to date, because no tool is available for the systematic evaluation of restriction enzyme combinations that can enrich for certain sites of interest in a genome. Herein, we present customised Reduced Representation Bisulfite Sequencing (cuRRBS), a novel and easy-to-use computational method that solves this problem. By computing the optimal enzymatic digestions and size selection steps required, cuRRBS generalises the traditional MspI-based Reduced Representation Bisulfite Sequencing (RRBS) protocol to all restriction enzyme combinations. In addition, cuRRBS estimates the fold-reduction in sequencing costs and provides a robustness value for the personalised RRBS protocol, allowing users to tailor the protocol to their experimental needs. Moreover, we show in silico that cuRRBS-defined restriction enzymes consistently out-perform MspI digestion in many biological systems, considering both CpG and CHG contexts. Finally, we have validated the accuracy of cuRRBS predictions for single and double enzyme digestions using two independent experimental datasets. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. A confidence interval analysis of sampling effort, sequencing depth, and taxonomic resolution of fungal community ecology in the era of high-throughput sequencing.

    PubMed

    Oono, Ryoko

    2017-01-01

    High-throughput sequencing technology has helped microbial community ecologists explore ecological and evolutionary patterns at unprecedented scales. The benefits of a large sample size still typically outweigh that of greater sequencing depths per sample for accurate estimations of ecological inferences. However, excluding or not sequencing rare taxa may mislead the answers to the questions 'how and why are communities different?' This study evaluates the confidence intervals of ecological inferences from high-throughput sequencing data of foliar fungal endophytes as case studies through a range of sampling efforts, sequencing depths, and taxonomic resolutions to understand how technical and analytical practices may affect our interpretations. Increasing sampling size reliably decreased confidence intervals across multiple community comparisons. However, the effects of sequencing depths on confidence intervals depended on how rare taxa influenced the dissimilarity estimates among communities and did not significantly decrease confidence intervals for all community comparisons. A comparison of simulated communities under random drift suggests that sequencing depths are important in estimating dissimilarities between microbial communities under neutral selective processes. Confidence interval analyses reveal important biases as well as biological trends in microbial community studies that otherwise may be ignored when communities are only compared for statistically significant differences.

  19. All-atom 3D structure prediction of transmembrane β-barrel proteins from sequences.

    PubMed

    Hayat, Sikander; Sander, Chris; Marks, Debora S; Elofsson, Arne

    2015-04-28

    Transmembrane β-barrels (TMBs) carry out major functions in substrate transport and protein biogenesis but experimental determination of their 3D structure is challenging. Encouraged by successful de novo 3D structure prediction of globular and α-helical membrane proteins from sequence alignments alone, we developed an approach to predict the 3D structure of TMBs. The approach combines the maximum-entropy evolutionary coupling method for predicting residue contacts (EVfold) with a machine-learning approach (boctopus2) for predicting β-strands in the barrel. In a blinded test for 19 TMB proteins of known structure that have a sufficient number of diverse homologous sequences available, this combined method (EVfold_bb) predicts hydrogen-bonded residue pairs between adjacent β-strands at an accuracy of ∼70%. This accuracy is sufficient for the generation of all-atom 3D models. In the transmembrane barrel region, the average 3D structure accuracy [template-modeling (TM) score] of top-ranked models is 0.54 (ranging from 0.36 to 0.85), with a higher (44%) number of residue pairs in correct strand-strand registration than in earlier methods (18%). Although the nonbarrel regions are predicted less accurately overall, the evolutionary couplings identify some highly constrained loop residues and, for FecA protein, the barrel including the structure of a plug domain can be accurately modeled (TM score = 0.68). Lower prediction accuracy tends to be associated with insufficient sequence information and we therefore expect increasing numbers of β-barrel families to become accessible to accurate 3D structure prediction as the number of available sequences increases.

  20. Systematic and stochastic influences on the performance of the MinION nanopore sequencer across a range of nucleotide bias

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

    Krishnakumar, Raga; Sinha, Anupama; Bird, Sara W.

    Emerging sequencing technologies are allowing us to characterize environmental, clinical and laboratory samples with increasing speed and detail, including real-time analysis and interpretation of data. One example of this is being able to rapidly and accurately detect a wide range of pathogenic organisms, both in the clinic and the field. Genomes can have radically different GC content however, such that accurate sequence analysis can be challenging depending upon the technology used. Here, we have characterized the performance of the Oxford MinION nanopore sequencer for detection and evaluation of organisms with a range of genomic nucleotide bias. We have diagnosed themore » quality of base-calling across individual reads and discovered that the position within the read affects base-calling and quality scores. Finally, we have evaluated the performance of the current state-of-the-art neural network-based MinION basecaller, characterizing its behavior with respect to systemic errors as well as context- and sequence-specific errors. Overall, we present a detailed characterization the capabilities of the MinION in terms of generating high-accuracy sequence data from genomes with a wide range of nucleotide content. This study provides a framework for designing the appropriate experiments that are the likely to lead to accurate and rapid field-forward diagnostics.« less

  1. Systematic and stochastic influences on the performance of the MinION nanopore sequencer across a range of nucleotide bias

    DOE PAGES

    Krishnakumar, Raga; Sinha, Anupama; Bird, Sara W.; ...

    2018-02-16

    Emerging sequencing technologies are allowing us to characterize environmental, clinical and laboratory samples with increasing speed and detail, including real-time analysis and interpretation of data. One example of this is being able to rapidly and accurately detect a wide range of pathogenic organisms, both in the clinic and the field. Genomes can have radically different GC content however, such that accurate sequence analysis can be challenging depending upon the technology used. Here, we have characterized the performance of the Oxford MinION nanopore sequencer for detection and evaluation of organisms with a range of genomic nucleotide bias. We have diagnosed themore » quality of base-calling across individual reads and discovered that the position within the read affects base-calling and quality scores. Finally, we have evaluated the performance of the current state-of-the-art neural network-based MinION basecaller, characterizing its behavior with respect to systemic errors as well as context- and sequence-specific errors. Overall, we present a detailed characterization the capabilities of the MinION in terms of generating high-accuracy sequence data from genomes with a wide range of nucleotide content. This study provides a framework for designing the appropriate experiments that are the likely to lead to accurate and rapid field-forward diagnostics.« less

  2. Genome sequencing and analysis of Yersina pestis KIM D27, an avirulent strain exempt from select agent regulation.

    PubMed

    Losada, Liliana; Varga, John J; Hostetler, Jessica; Radune, Diana; Kim, Maria; Durkin, Scott; Schneewind, Olaf; Nierman, William C

    2011-04-29

    Yersinia pestis is the causative agent of the plague. Y. pestis KIM 10+ strain was passaged and selected for loss of the 102 kb pgm locus, resulting in an attenuated strain, KIM D27. In this study, whole genome sequencing was performed on KIM D27 in order to identify any additional differences. Initial assemblies of 454 data were highly fragmented, and various bioinformatic tools detected between 15 and 465 SNPs and INDELs when comparing both strains, the vast majority associated with A or T homopolymer sequences. Consequently, Illumina sequencing was performed to improve the quality of the assembly. Hybrid sequence assemblies were performed and a total of 56 validated SNP/INDELs and 5 repeat differences were identified in the D27 strain relative to published KIM 10+ sequence. However, further analysis showed that 55 of these SNP/INDELs and 3 repeats were errors in the KIM 10+ reference sequence. We conclude that both 454 and Illumina sequencing were required to obtain the most accurate and rapid sequence results for Y. pestis KIMD27. SNP and INDELS calls were most accurate when both Newbler and CLC Genomics Workbench were employed. For purposes of obtaining high quality genome sequence differences between strains, any identified differences should be verified in both the new and reference genomes.

  3. VarDict: a novel and versatile variant caller for next-generation sequencing in cancer research

    PubMed Central

    Lai, Zhongwu; Markovets, Aleksandra; Ahdesmaki, Miika; Chapman, Brad; Hofmann, Oliver; McEwen, Robert; Johnson, Justin; Dougherty, Brian; Barrett, J. Carl; Dry, Jonathan R.

    2016-01-01

    Abstract Accurate variant calling in next generation sequencing (NGS) is critical to understand cancer genomes better. Here we present VarDict, a novel and versatile variant caller for both DNA- and RNA-sequencing data. VarDict simultaneously calls SNV, MNV, InDels, complex and structural variants, expanding the detected genetic driver landscape of tumors. It performs local realignments on the fly for more accurate allele frequency estimation. VarDict performance scales linearly to sequencing depth, enabling ultra-deep sequencing used to explore tumor evolution or detect tumor DNA circulating in blood. In addition, VarDict performs amplicon aware variant calling for polymerase chain reaction (PCR)-based targeted sequencing often used in diagnostic settings, and is able to detect PCR artifacts. Finally, VarDict also detects differences in somatic and loss of heterozygosity variants between paired samples. VarDict reprocessing of The Cancer Genome Atlas (TCGA) Lung Adenocarcinoma dataset called known driver mutations in KRAS, EGFR, BRAF, PIK3CA and MET in 16% more patients than previously published variant calls. We believe VarDict will greatly facilitate application of NGS in clinical cancer research. PMID:27060149

  4. 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

  5. Copy number variants calling for single cell sequencing data by multi-constrained optimization.

    PubMed

    Xu, Bo; Cai, Hongmin; Zhang, Changsheng; Yang, Xi; Han, Guoqiang

    2016-08-01

    Variations in DNA copy number carry important information on genome evolution and regulation of DNA replication in cancer cells. The rapid development of single-cell sequencing technology allows one to explore gene expression heterogeneity among single-cells, thus providing important cancer cell evolution information. Single-cell DNA/RNA sequencing data usually have low genome coverage, which requires an extra step of amplification to accumulate enough samples. However, such amplification will introduce large bias and makes bioinformatics analysis challenging. Accurately modeling the distribution of sequencing data and effectively suppressing the bias influence is the key to success variations analysis. Recent advances demonstrate the technical noises by amplification are more likely to follow negative binomial distribution, a special case of Poisson distribution. Thus, we tackle the problem CNV detection by formulating it into a quadratic optimization problem involving two constraints, in which the underling signals are corrupted by Poisson distributed noises. By imposing the constraints of sparsity and smoothness, the reconstructed read depth signals from single-cell sequencing data are anticipated to fit the CNVs patterns more accurately. An efficient numerical solution based on the classical alternating direction minimization method (ADMM) is tailored to solve the proposed model. We demonstrate the advantages of the proposed method using both synthetic and empirical single-cell sequencing data. Our experimental results demonstrate that the proposed method achieves excellent performance and high promise of success with single-cell sequencing data. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  6. A Model of BGA Thermal Fatigue Life Prediction Considering Load Sequence Effects

    PubMed Central

    Hu, Weiwei; Li, Yaqiu; Sun, Yufeng; Mosleh, Ali

    2016-01-01

    Accurate testing history data is necessary for all fatigue life prediction approaches, but such data is always deficient especially for the microelectronic devices. Additionally, the sequence of the individual load cycle plays an important role in physical fatigue damage. However, most of the existing models based on the linear damage accumulation rule ignore the sequence effects. This paper proposes a thermal fatigue life prediction model for ball grid array (BGA) packages to take into consideration the load sequence effects. For the purpose of improving the availability and accessibility of testing data, a new failure criterion is discussed and verified by simulation and experimentation. The consequences for the fatigue underlying sequence load conditions are shown. PMID:28773980

  7. Chiron: translating nanopore raw signal directly into nucleotide sequence using deep learning.

    PubMed

    Teng, Haotian; Cao, Minh Duc; Hall, Michael B; Duarte, Tania; Wang, Sheng; Coin, Lachlan J M

    2018-05-01

    Sequencing by translocating DNA fragments through an array of nanopores is a rapidly maturing technology that offers faster and cheaper sequencing than other approaches. However, accurately deciphering the DNA sequence from the noisy and complex electrical signal is challenging. Here, we report Chiron, the first deep learning model to achieve end-to-end basecalling and directly translate the raw signal to DNA sequence without the error-prone segmentation step. Trained with only a small set of 4,000 reads, we show that our model provides state-of-the-art basecalling accuracy, even on previously unseen species. Chiron achieves basecalling speeds of more than 2,000 bases per second using desktop computer graphics processing units.

  8. Anatomy of a hash-based long read sequence mapping algorithm for next generation DNA sequencing.

    PubMed

    Misra, Sanchit; Agrawal, Ankit; Liao, Wei-keng; Choudhary, Alok

    2011-01-15

    Recently, a number of programs have been proposed for mapping short reads to a reference genome. Many of them are heavily optimized for short-read mapping and hence are very efficient for shorter queries, but that makes them inefficient or not applicable for reads longer than 200 bp. However, many sequencers are already generating longer reads and more are expected to follow. For long read sequence mapping, there are limited options; BLAT, SSAHA2, FANGS and BWA-SW are among the popular ones. However, resequencing and personalized medicine need much faster software to map these long sequencing reads to a reference genome to identify SNPs or rare transcripts. We present AGILE (AliGnIng Long rEads), a hash table based high-throughput sequence mapping algorithm for longer 454 reads that uses diagonal multiple seed-match criteria, customized q-gram filtering and a dynamic incremental search approach among other heuristics to optimize every step of the mapping process. In our experiments, we observe that AGILE is more accurate than BLAT, and comparable to BWA-SW and SSAHA2. For practical error rates (< 5%) and read lengths (200-1000 bp), AGILE is significantly faster than BLAT, SSAHA2 and BWA-SW. Even for the other cases, AGILE is comparable to BWA-SW and several times faster than BLAT and SSAHA2. http://www.ece.northwestern.edu/~smi539/agile.html.

  9. Genetic Perturbation of the Maize Methylome[W

    PubMed Central

    Li, Qing; Hermanson, Peter J.; Zaunbrecher, Virginia M.; Song, Jawon; Wendt, Jennifer; Rosenbaum, Heidi; Madzima, Thelma F.; Sloan, Amy E.; Huang, Ji; Burgess, Daniel L.; Richmond, Todd A.; McGinnis, Karen M.; Meeley, Robert B.; Danilevskaya, Olga N.; Vaughn, Matthew W.; Kaeppler, Shawn M.; Jeddeloh, Jeffrey A.

    2014-01-01

    DNA methylation can play important roles in the regulation of transposable elements and genes. A collection of mutant alleles for 11 maize (Zea mays) genes predicted to play roles in controlling DNA methylation were isolated through forward- or reverse-genetic approaches. Low-coverage whole-genome bisulfite sequencing and high-coverage sequence-capture bisulfite sequencing were applied to mutant lines to determine context- and locus-specific effects of these mutations on DNA methylation profiles. Plants containing mutant alleles for components of the RNA-directed DNA methylation pathway exhibit loss of CHH methylation at many loci as well as CG and CHG methylation at a small number of loci. Plants containing loss-of-function alleles for chromomethylase (CMT) genes exhibit strong genome-wide reductions in CHG methylation and some locus-specific loss of CHH methylation. In an attempt to identify stocks with stronger reductions in DNA methylation levels than provided by single gene mutations, we performed crosses to create double mutants for the maize CMT3 orthologs, Zmet2 and Zmet5, and for the maize DDM1 orthologs, Chr101 and Chr106. While loss-of-function alleles are viable as single gene mutants, the double mutants were not recovered, suggesting that severe perturbations of the maize methylome may have stronger deleterious phenotypic effects than in Arabidopsis thaliana. PMID:25527708

  10. COBRA-Seq: Sensitive and Quantitative Methylome Profiling

    PubMed Central

    Varinli, Hilal; Statham, Aaron L.; Clark, Susan J.; Molloy, Peter L.; Ross, Jason P.

    2015-01-01

    Combined Bisulfite Restriction Analysis (COBRA) quantifies DNA methylation at a specific locus. It does so via digestion of PCR amplicons produced from bisulfite-treated DNA, using a restriction enzyme that contains a cytosine within its recognition sequence, such as TaqI. Here, we introduce COBRA-seq, a genome wide reduced methylome method that requires minimal DNA input (0.1–1.0 μg) and can either use PCR or linear amplification to amplify the sequencing library. Variants of COBRA-seq can be used to explore CpG-depleted as well as CpG-rich regions in vertebrate DNA. The choice of enzyme influences enrichment for specific genomic features, such as CpG-rich promoters and CpG islands, or enrichment for less CpG dense regions such as enhancers. COBRA-seq coupled with linear amplification has the additional advantage of reduced PCR bias by producing full length fragments at high abundance. Unlike other reduced representative methylome methods, COBRA-seq has great flexibility in the choice of enzyme and can be multiplexed and tuned, to reduce sequencing costs and to interrogate different numbers of sites. Moreover, COBRA-seq is applicable to non-model organisms without the reference genome and compatible with the investigation of non-CpG methylation by using restriction enzymes containing CpA, CpT, and CpC in their recognition site. PMID:26512698

  11. Massively Parallel Sequencing Detected a Mutation in the MFN2 Gene Missed by Sanger Sequencing Due to a Primer Mismatch on an SNP Site.

    PubMed

    Neupauerová, Jana; Grečmalová, Dagmar; Seeman, Pavel; Laššuthová, Petra

    2016-05-01

    We describe a patient with early onset severe axonal Charcot-Marie-Tooth disease (CMT2) with dominant inheritance, in whom Sanger sequencing failed to detect a mutation in the mitofusin 2 (MFN2) gene because of a single nucleotide polymorphism (rs2236057) under the PCR primer sequence. The severe early onset phenotype and the family history with severely affected mother (died after delivery) was very suggestive of CMT2A and this suspicion was finally confirmed by a MFN2 mutation. The mutation p.His361Tyr was later detected in the patient by massively parallel sequencing with a gene panel for hereditary neuropathies. According to this information, new primers for amplification and sequencing were designed which bind away from the polymorphic sites of the patient's DNA. Sanger sequencing with these new primers then confirmed the heterozygous mutation in the MFN2 gene in this patient. This case report shows that massively parallel sequencing may in some rare cases be more sensitive than Sanger sequencing and highlights the importance of accurate primer design which requires special attention. © 2016 John Wiley & Sons Ltd/University College London.

  12. TCRmodel: high resolution modeling of T cell receptors from sequence.

    PubMed

    Gowthaman, Ragul; Pierce, Brian G

    2018-05-22

    T cell receptors (TCRs), along with antibodies, are responsible for specific antigen recognition in the adaptive immune response, and millions of unique TCRs are estimated to be present in each individual. Understanding the structural basis of TCR targeting has implications in vaccine design, autoimmunity, as well as T cell therapies for cancer. Given advances in deep sequencing leading to immune repertoire-level TCR sequence data, fast and accurate modeling methods are needed to elucidate shared and unique 3D structural features of these molecules which lead to their antigen targeting and cross-reactivity. We developed a new algorithm in the program Rosetta to model TCRs from sequence, and implemented this functionality in a web server, TCRmodel. This web server provides an easy to use interface, and models are generated quickly that users can investigate in the browser and download. Benchmarking of this method using a set of nonredundant recently released TCR crystal structures shows that models are accurate and compare favorably to models from another available modeling method. This server enables the community to obtain insights into TCRs of interest, and can be combined with methods to model and design TCR recognition of antigens. The TCRmodel server is available at: http://tcrmodel.ibbr.umd.edu/.

  13. Integrative structural annotation of de novo RNA-Seq provides an accurate reference gene set of the enormous genome of the onion (Allium cepa L.).

    PubMed

    Kim, Seungill; Kim, Myung-Shin; Kim, Yong-Min; Yeom, Seon-In; Cheong, Kyeongchae; Kim, Ki-Tae; Jeon, Jongbum; Kim, Sunggil; Kim, Do-Sun; Sohn, Seong-Han; Lee, Yong-Hwan; Choi, Doil

    2015-02-01

    The onion (Allium cepa L.) is one of the most widely cultivated and consumed vegetable crops in the world. Although a considerable amount of onion transcriptome data has been deposited into public databases, the sequences of the protein-coding genes are not accurate enough to be used, owing to non-coding sequences intermixed with the coding sequences. We generated a high-quality, annotated onion transcriptome from de novo sequence assembly and intensive structural annotation using the integrated structural gene annotation pipeline (ISGAP), which identified 54,165 protein-coding genes among 165,179 assembled transcripts totalling 203.0 Mb by eliminating the intron sequences. ISGAP performed reliable annotation, recognizing accurate gene structures based on reference proteins, and ab initio gene models of the assembled transcripts. Integrative functional annotation and gene-based SNP analysis revealed a whole biological repertoire of genes and transcriptomic variation in the onion. The method developed in this study provides a powerful tool for the construction of reference gene sets for organisms based solely on de novo transcriptome data. Furthermore, the reference genes and their variation described here for the onion represent essential tools for molecular breeding and gene cloning in Allium spp. © The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  14. Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

    PubMed

    Andréasson, H; Asp, A; Alderborn, A; Gyllensten, U; Allen, M

    2002-01-01

    Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

  15. Combining Structural Modeling with Ensemble Machine Learning to Accurately Predict Protein Fold Stability and Binding Affinity Effects upon Mutation

    PubMed Central

    Garcia Lopez, Sebastian; Kim, Philip M.

    2014-01-01

    Advances in sequencing have led to a rapid accumulation of mutations, some of which are associated with diseases. However, to draw mechanistic conclusions, a biochemical understanding of these mutations is necessary. For coding mutations, accurate prediction of significant changes in either the stability of proteins or their affinity to their binding partners is required. Traditional methods have used semi-empirical force fields, while newer methods employ machine learning of sequence and structural features. Here, we show how combining both of these approaches leads to a marked boost in accuracy. We introduce ELASPIC, a novel ensemble machine learning approach that is able to predict stability effects upon mutation in both, domain cores and domain-domain interfaces. We combine semi-empirical energy terms, sequence conservation, and a wide variety of molecular details with a Stochastic Gradient Boosting of Decision Trees (SGB-DT) algorithm. The accuracy of our predictions surpasses existing methods by a considerable margin, achieving correlation coefficients of 0.77 for stability, and 0.75 for affinity predictions. Notably, we integrated homology modeling to enable proteome-wide prediction and show that accurate prediction on modeled structures is possible. Lastly, ELASPIC showed significant differences between various types of disease-associated mutations, as well as between disease and common neutral mutations. Unlike pure sequence-based prediction methods that try to predict phenotypic effects of mutations, our predictions unravel the molecular details governing the protein instability, and help us better understand the molecular causes of diseases. PMID:25243403

  16. BPP: a sequence-based algorithm for branch point prediction.

    PubMed

    Zhang, Qing; Fan, Xiaodan; Wang, Yejun; Sun, Ming-An; Shao, Jianlin; Guo, Dianjing

    2017-10-15

    Although high-throughput sequencing methods have been proposed to identify splicing branch points in the human genome, these methods can only detect a small fraction of the branch points subject to the sequencing depth, experimental cost and the expression level of the mRNA. An accurate computational model for branch point prediction is therefore an ongoing objective in human genome research. We here propose a novel branch point prediction algorithm that utilizes information on the branch point sequence and the polypyrimidine tract. Using experimentally validated data, we demonstrate that our proposed method outperforms existing methods. Availability and implementation: https://github.com/zhqingit/BPP. djguo@cuhk.edu.hk. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  17. VarDetect: a nucleotide sequence variation exploratory tool

    PubMed Central

    Ngamphiw, Chumpol; Kulawonganunchai, Supasak; Assawamakin, Anunchai; Jenwitheesuk, Ekachai; Tongsima, Sissades

    2008-01-01

    Background Single nucleotide polymorphisms (SNPs) are the most commonly studied units of genetic variation. The discovery of such variation may help to identify causative gene mutations in monogenic diseases and SNPs associated with predisposing genes in complex diseases. Accurate detection of SNPs requires software that can correctly interpret chromatogram signals to nucleotides. Results We present VarDetect, a stand-alone nucleotide variation exploratory tool that automatically detects nucleotide variation from fluorescence based chromatogram traces. Accurate SNP base-calling is achieved using pre-calculated peak content ratios, and is enhanced by rules which account for common sequence reading artifacts. The proposed software tool is benchmarked against four other well-known SNP discovery software tools (PolyPhred, novoSNP, Genalys and Mutation Surveyor) using fluorescence based chromatograms from 15 human genes. These chromatograms were obtained from sequencing 16 two-pooled DNA samples; a total of 32 individual DNA samples. In this comparison of automatic SNP detection tools, VarDetect achieved the highest detection efficiency. Availability VarDetect is compatible with most major operating systems such as Microsoft Windows, Linux, and Mac OSX. The current version of VarDetect is freely available at . PMID:19091032

  18. Infrared thermal facial image sequence registration analysis and verification

    NASA Astrophysics Data System (ADS)

    Chen, Chieh-Li; Jian, Bo-Lin

    2015-03-01

    To study the emotional responses of subjects to the International Affective Picture System (IAPS), infrared thermal facial image sequence is preprocessed for registration before further analysis such that the variance caused by minor and irregular subject movements is reduced. Without affecting the comfort level and inducing minimal harm, this study proposes an infrared thermal facial image sequence registration process that will reduce the deviations caused by the unconscious head shaking of the subjects. A fixed image for registration is produced through the localization of the centroid of the eye region as well as image translation and rotation processes. Thermal image sequencing will then be automatically registered using the two-stage genetic algorithm proposed. The deviation before and after image registration will be demonstrated by image quality indices. The results show that the infrared thermal image sequence registration process proposed in this study is effective in localizing facial images accurately, which will be beneficial to the correlation analysis of psychological information related to the facial area.

  19. Integration of Temporal and Ordinal Information During Serial Interception Sequence Learning

    PubMed Central

    Gobel, Eric W.; Sanchez, Daniel J.; Reber, Paul J.

    2011-01-01

    The expression of expert motor skills typically involves learning to perform a precisely timed sequence of movements (e.g., language production, music performance, athletic skills). Research examining incidental sequence learning has previously relied on a perceptually-cued task that gives participants exposure to repeating motor sequences but does not require timing of responses for accuracy. Using a novel perceptual-motor sequence learning task, learning a precisely timed cued sequence of motor actions is shown to occur without explicit instruction. Participants learned a repeating sequence through practice and showed sequence-specific knowledge via a performance decrement when switched to an unfamiliar sequence. In a second experiment, the integration of representation of action order and timing sequence knowledge was examined. When either action order or timing sequence information was selectively disrupted, performance was reduced to levels similar to completely novel sequences. Unlike prior sequence-learning research that has found timing information to be secondary to learning action sequences, when the task demands require accurate action and timing information, an integrated representation of these types of information is acquired. These results provide the first evidence for incidental learning of fully integrated action and timing sequence information in the absence of an independent representation of action order, and suggest that this integrative mechanism may play a material role in the acquisition of complex motor skills. PMID:21417511

  20. Are commercial providers a viable option for clinical bacterial sequencing?

    PubMed

    Raven, Kathy; Blane, Beth; Churcher, Carol; Parkhill, Julian; Peacock, Sharon J

    2018-04-05

    Bacterial whole-genome sequencing in the clinical setting has the potential to bring major improvements to infection control and clinical practice. Sequencing instruments are not currently available in the majority of routine microbiology laboratories worldwide, but an alternative is to use external sequencing providers. To foster discussion around this we investigated whether send-out services were a viable option. Four providers offering MiSeq sequencing were selected based on cost and evaluated based on the service provided and sequence data quality. DNA was prepared from five methicillin-resistant Staphylococcus aureus (MRSA) isolates, four of which were investigated during a previously published outbreak in the UK together with a reference MRSA isolate (ST22 HO 5096 0412). Cost of sequencing per isolate ranged from £155 to £342 and turnaround times from DNA postage to arrival of sequence data ranged from 12 to 63 days. Comparison of commercially generated genomes against the original sequence data demonstrated very high concordance, with no more than one single nucleotide polymorphism (SNP) difference on core genome mapping between the original sequences and the new sequence for all four providers. Multilocus sequence type could not be assigned based on assembly for the two cheapest sequence providers due to fragmented assemblies probably caused by a lower output of sequence data per isolate. Our results indicate that external providers returned highly accurate genome data, but that improvements are required in turnaround time to make this a viable option for use in clinical practice.

  1. Effective Feature Selection for Classification of Promoter Sequences.

    PubMed

    K, Kouser; P G, Lavanya; Rangarajan, Lalitha; K, Acharya Kshitish

    2016-01-01

    Exploring novel computational methods in making sense of biological data has not only been a necessity, but also productive. A part of this trend is the search for more efficient in silico methods/tools for analysis of promoters, which are parts of DNA sequences that are involved in regulation of expression of genes into other functional molecules. Promoter regions vary greatly in their function based on the sequence of nucleotides and the arrangement of protein-binding short-regions called motifs. In fact, the regulatory nature of the promoters seems to be largely driven by the selective presence and/or the arrangement of these motifs. Here, we explore computational classification of promoter sequences based on the pattern of motif distributions, as such classification can pave a new way of functional analysis of promoters and to discover the functionally crucial motifs. We make use of Position Specific Motif Matrix (PSMM) features for exploring the possibility of accurately classifying promoter sequences using some of the popular classification techniques. The classification results on the complete feature set are low, perhaps due to the huge number of features. We propose two ways of reducing features. Our test results show improvement in the classification output after the reduction of features. The results also show that decision trees outperform SVM (Support Vector Machine), KNN (K Nearest Neighbor) and ensemble classifier LibD3C, particularly with reduced features. The proposed feature selection methods outperform some of the popular feature transformation methods such as PCA and SVD. Also, the methods proposed are as accurate as MRMR (feature selection method) but much faster than MRMR. Such methods could be useful to categorize new promoters and explore regulatory mechanisms of gene expressions in complex eukaryotic species.

  2. A confidence interval analysis of sampling effort, sequencing depth, and taxonomic resolution of fungal community ecology in the era of high-throughput sequencing

    PubMed Central

    2017-01-01

    High-throughput sequencing technology has helped microbial community ecologists explore ecological and evolutionary patterns at unprecedented scales. The benefits of a large sample size still typically outweigh that of greater sequencing depths per sample for accurate estimations of ecological inferences. However, excluding or not sequencing rare taxa may mislead the answers to the questions ‘how and why are communities different?’ This study evaluates the confidence intervals of ecological inferences from high-throughput sequencing data of foliar fungal endophytes as case studies through a range of sampling efforts, sequencing depths, and taxonomic resolutions to understand how technical and analytical practices may affect our interpretations. Increasing sampling size reliably decreased confidence intervals across multiple community comparisons. However, the effects of sequencing depths on confidence intervals depended on how rare taxa influenced the dissimilarity estimates among communities and did not significantly decrease confidence intervals for all community comparisons. A comparison of simulated communities under random drift suggests that sequencing depths are important in estimating dissimilarities between microbial communities under neutral selective processes. Confidence interval analyses reveal important biases as well as biological trends in microbial community studies that otherwise may be ignored when communities are only compared for statistically significant differences. PMID:29253889

  3. Statistical processing of large image sequences.

    PubMed

    Khellah, F; Fieguth, P; Murray, M J; Allen, M

    2005-01-01

    The dynamic estimation of large-scale stochastic image sequences, as frequently encountered in remote sensing, is important in a variety of scientific applications. However, the size of such images makes conventional dynamic estimation methods, for example, the Kalman and related filters, impractical. In this paper, we present an approach that emulates the Kalman filter, but with considerably reduced computational and storage requirements. Our approach is illustrated in the context of a 512 x 512 image sequence of ocean surface temperature. The static estimation step, the primary contribution here, uses a mixture of stationary models to accurately mimic the effect of a nonstationary prior, simplifying both computational complexity and modeling. Our approach provides an efficient, stable, positive-definite model which is consistent with the given correlation structure. Thus, the methods of this paper may find application in modeling and single-frame estimation.

  4. Genome Sequencing and Analysis of Yersina pestis KIM D27, an Avirulent Strain Exempt from Select Agent Regulation

    PubMed Central

    Losada, Liliana; Varga, John J.; Hostetler, Jessica; Radune, Diana; Kim, Maria; Durkin, Scott; Schneewind, Olaf; Nierman, William C.

    2011-01-01

    Yersinia pestis is the causative agent of the plague. Y. pestis KIM 10+ strain was passaged and selected for loss of the 102 kb pgm locus, resulting in an attenuated strain, KIM D27. In this study, whole genome sequencing was performed on KIM D27 in order to identify any additional differences. Initial assemblies of 454 data were highly fragmented, and various bioinformatic tools detected between 15 and 465 SNPs and INDELs when comparing both strains, the vast majority associated with A or T homopolymer sequences. Consequently, Illumina sequencing was performed to improve the quality of the assembly. Hybrid sequence assemblies were performed and a total of 56 validated SNP/INDELs and 5 repeat differences were identified in the D27 strain relative to published KIM 10+ sequence. However, further analysis showed that 55 of these SNP/INDELs and 3 repeats were errors in the KIM 10+ reference sequence. We conclude that both 454 and Illumina sequencing were required to obtain the most accurate and rapid sequence results for Y. pestis KIMD27. SNP and INDELS calls were most accurate when both Newbler and CLC Genomics Workbench were employed. For purposes of obtaining high quality genome sequence differences between strains, any identified differences should be verified in both the new and reference genomes. PMID:21559501

  5. Methylation levels of the "long interspersed nucleotide element-1" repetitive sequences predict survival of melanoma patients.

    PubMed

    Sigalotti, Luca; Fratta, Elisabetta; Bidoli, Ettore; Covre, Alessia; Parisi, Giulia; Colizzi, Francesca; Coral, Sandra; Massarut, Samuele; Kirkwood, John M; Maio, Michele

    2011-05-26

    The prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established. Genome-wide alterations in DNA methylation are a common event in cancer. This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients. Overall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients. The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis. Hypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association. Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis. Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively. The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences. Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01). LINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC. Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients.

  6. Methylation levels of the "long interspersed nucleotide element-1" repetitive sequences predict survival of melanoma patients

    PubMed Central

    2011-01-01

    Background The prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established. Genome-wide alterations in DNA methylation are a common event in cancer. This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients. Methods Overall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients. The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis. Results Hypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association. Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis. Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively. The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences. Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01). Conclusion LINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC. Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients. PMID:21615918

  7. Sequence2Vec: a novel embedding approach for modeling transcription factor binding affinity landscape.

    PubMed

    Dai, Hanjun; Umarov, Ramzan; Kuwahara, Hiroyuki; Li, Yu; Song, Le; Gao, Xin

    2017-11-15

    An accurate characterization of transcription factor (TF)-DNA affinity landscape is crucial to a quantitative understanding of the molecular mechanisms underpinning endogenous gene regulation. While recent advances in biotechnology have brought the opportunity for building binding affinity prediction methods, the accurate characterization of TF-DNA binding affinity landscape still remains a challenging problem. Here we propose a novel sequence embedding approach for modeling the transcription factor binding affinity landscape. Our method represents DNA binding sequences as a hidden Markov model which captures both position specific information and long-range dependency in the sequence. A cornerstone of our method is a novel message passing-like embedding algorithm, called Sequence2Vec, which maps these hidden Markov models into a common nonlinear feature space and uses these embedded features to build a predictive model. Our method is a novel combination of the strength of probabilistic graphical models, feature space embedding and deep learning. We conducted comprehensive experiments on over 90 large-scale TF-DNA datasets which were measured by different high-throughput experimental technologies. Sequence2Vec outperforms alternative machine learning methods as well as the state-of-the-art binding affinity prediction methods. Our program is freely available at https://github.com/ramzan1990/sequence2vec. xin.gao@kaust.edu.sa or lsong@cc.gatech.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

  8. Ultra-deep mutant spectrum profiling: improving sequencing accuracy using overlapping read pairs.

    PubMed

    Chen-Harris, Haiyin; Borucki, Monica K; Torres, Clinton; Slezak, Tom R; Allen, Jonathan E

    2013-02-12

    High throughput sequencing is beginning to make a transformative impact in the area of viral evolution. Deep sequencing has the potential to reveal the mutant spectrum within a viral sample at high resolution, thus enabling the close examination of viral mutational dynamics both within- and between-hosts. The challenge however, is to accurately model the errors in the sequencing data and differentiate real viral mutations, particularly those that exist at low frequencies, from sequencing errors. We demonstrate that overlapping read pairs (ORP) -- generated by combining short fragment sequencing libraries and longer sequencing reads -- significantly reduce sequencing error rates and improve rare variant detection accuracy. Using this sequencing protocol and an error model optimized for variant detection, we are able to capture a large number of genetic mutations present within a viral population at ultra-low frequency levels (<0.05%). Our rare variant detection strategies have important implications beyond viral evolution and can be applied to any basic and clinical research area that requires the identification of rare mutations.

  9. Application of discrete Fourier inter-coefficient difference for assessing genetic sequence similarity.

    PubMed

    King, Brian R; Aburdene, Maurice; Thompson, Alex; Warres, Zach

    2014-01-01

    Digital signal processing (DSP) techniques for biological sequence analysis continue to grow in popularity due to the inherent digital nature of these sequences. DSP methods have demonstrated early success for detection of coding regions in a gene. Recently, these methods are being used to establish DNA gene similarity. We present the inter-coefficient difference (ICD) transformation, a novel extension of the discrete Fourier transformation, which can be applied to any DNA sequence. The ICD method is a mathematical, alignment-free DNA comparison method that generates a genetic signature for any DNA sequence that is used to generate relative measures of similarity among DNA sequences. We demonstrate our method on a set of insulin genes obtained from an evolutionarily wide range of species, and on a set of avian influenza viral sequences, which represents a set of highly similar sequences. We compare phylogenetic trees generated using our technique against trees generated using traditional alignment techniques for similarity and demonstrate that the ICD method produces a highly accurate tree without requiring an alignment prior to establishing sequence similarity.

  10. Rapid-Sequence Serial Sexual Homicides.

    PubMed

    Schlesinger, Louis B; Ramirez, Stephanie; Tusa, Brittany; Jarvis, John P; Erdberg, Philip

    2017-03-01

    Serial sexual murderers have been described as committing homicides in a methodical manner, taking substantial time between offenses to elude the authorities. The results of our study of the temporal patterns (i.e., the length of time between homicides) of a nonrandom national sample of 44 serial sexual murderers and their 201 victims indicate that this representation may not always be accurate. Although 25 offenders (56.8%) killed with longer than a 14-day period between homicides, a sizeable subgroup was identified: 19 offenders (43.2%) who committed homicides in rapid-sequence fashion, with fewer than 14 days between all or some of the murders. Six offenders (13.6%) killed all their victims in one rapid-sequence, spree-like episode, with homicides just days apart or sometimes two murders in the same day. Thirteen offenders (29.5%) killed in one or two rapid-sequence clusters (i.e., more than one murder within a 14-day period, as well as additional homicides with greater than 14 days between each). The purpose of our study was to describe this subgroup of rapid-sequence offenders who have not been identified until now. These findings argue for accelerated forensic assessments of dangerousness and public safety when a sexual murder is detected. Psychiatric disorders with rapidly occurring symptom patterns, or even atypical mania or mood dysregulation, may serve as exemplars for understanding this extraordinary group of offenders. © 2017 American Academy of Psychiatry and the Law.

  11. Accurate clinical genetic testing for autoinflammatory diseases using the next-generation sequencing platform MiSeq.

    PubMed

    Nakayama, Manabu; Oda, Hirotsugu; Nakagawa, Kenji; Yasumi, Takahiro; Kawai, Tomoki; Izawa, Kazushi; Nishikomori, Ryuta; Heike, Toshio; Ohara, Osamu

    2017-03-01

    Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program-which detects and avoids common SNPs in gene-specific PCR primers-we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.

  12. Allele-specific copy-number discovery from whole-genome and whole-exome sequencing

    PubMed Central

    Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J.; Szatkiewicz, Jin P.

    2015-01-01

    Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. PMID:25883151

  13. Transcriptome analysis by strand-specific sequencing of complementary DNA

    PubMed Central

    Parkhomchuk, Dmitri; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Banaru, Maria; Hallen, Linda; Krobitsch, Sylvia; Lehrach, Hans; Soldatov, Alexey

    2009-01-01

    High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online. PMID:19620212

  14. Transcriptome analysis by strand-specific sequencing of complementary DNA.

    PubMed

    Parkhomchuk, Dmitri; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Banaru, Maria; Hallen, Linda; Krobitsch, Sylvia; Lehrach, Hans; Soldatov, Alexey

    2009-10-01

    High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.

  15. Accurate measurement of heteronuclear dipolar couplings by phase-alternating R-symmetry (PARS) sequences in magic angle spinning NMR spectroscopy

    NASA Astrophysics Data System (ADS)

    Hou, Guangjin; Lu, Xingyu; Vega, Alexander J.; Polenova, Tatyana

    2014-09-01

    We report a Phase-Alternating R-Symmetry (PARS) dipolar recoupling scheme for accurate measurement of heteronuclear 1H-X (X = 13C, 15N, 31P, etc.) dipolar couplings in MAS NMR experiments. It is an improvement of conventional C- and R-symmetry type DIPSHIFT experiments where, in addition to the dipolar interaction, the 1H CSA interaction persists and thereby introduces considerable errors in the dipolar measurements. In PARS, phase-shifted RN symmetry pulse blocks applied on the 1H spins combined with π pulses applied on the X spins at the end of each RN block efficiently suppress the effect from 1H chemical shift anisotropy, while keeping the 1H-X dipolar couplings intact. Another advantage over conventional DIPSHIFT experiments, which require the signal to be detected in the form of a reduced-intensity Hahn echo, is that the series of π pulses refocuses the X chemical shift and avoids the necessity of echo formation. PARS permits determination of accurate dipolar couplings in a single experiment; it is suitable for a wide range of MAS conditions including both slow and fast MAS frequencies; and it assures dipolar truncation from the remote protons. The performance of PARS is tested on two model systems, [15N]-N-acetyl-valine and [U-13C,15N]-N-formyl-Met-Leu-Phe tripeptide. The application of PARS for site-resolved measurement of accurate 1H-15N dipolar couplings in the context of 3D experiments is presented on U-13C,15N-enriched dynein light chain protein LC8.

  16. ReQON: a Bioconductor package for recalibrating quality scores from next-generation sequencing data

    PubMed Central

    2012-01-01

    Background Next-generation sequencing technologies have become important tools for genome-wide studies. However, the quality scores that are assigned to each base have been shown to be inaccurate. If the quality scores are used in downstream analyses, these inaccuracies can have a significant impact on the results. Results Here we present ReQON, a tool that recalibrates the base quality scores from an input BAM file of aligned sequencing data using logistic regression. ReQON also generates diagnostic plots showing the effectiveness of the recalibration. We show that ReQON produces quality scores that are both more accurate, in the sense that they more closely correspond to the probability of a sequencing error, and do a better job of discriminating between sequencing errors and non-errors than the original quality scores. We also compare ReQON to other available recalibration tools and show that ReQON is less biased and performs favorably in terms of quality score accuracy. Conclusion ReQON is an open source software package, written in R and available through Bioconductor, for recalibrating base quality scores for next-generation sequencing data. ReQON produces a new BAM file with more accurate quality scores, which can improve the results of downstream analysis, and produces several diagnostic plots showing the effectiveness of the recalibration. PMID:22946927

  17. Effects of metal-rich particulate matter exposure on exogenous and endogenous viral sequence methylation in healthy steel-workers.

    PubMed

    Mercorio, Roberta; Bonzini, Matteo; Angelici, Laura; Iodice, Simona; Delbue, Serena; Mariani, Jacopo; Apostoli, Pietro; Pesatori, Angela Cecilia; Bollati, Valentina

    2017-11-01

    Inhaled particles have been shown to produce systemic changes in DNA methylation. Global hypomethylation has been associated to viral sequence reactivation, possibly linked to the activation of pro-inflammatory pathways occurring after exposure. This observation provides a rationale to investigate viral sequence (both exogenous and endogenous) methylation in association to metal-rich particulate matter exposure. To verify this hypothesis, we chose the Wp promoter of the Epstein-Barr Virus (EBV-Wp) and the promoter of the human-endogenous-retrovirus w (HERV-w), respectively as a paradigm of an exogenous and an endogenous retroviral sequence, to be investigated by bisulfite PCR Pyrosequencing. We enrolled 63 male workers in an electric furnace steel plant, exposed to high level of metal-rich particulate matter. Comparing samples obtained in the first day of a work week (time 0-baseline, after 2 days off work) and the samples obtained after 3 days of work (time 1-post exposure), the mean methylation of EBV-Wp was significantly higher at baseline compared to post-exposure (mean baseline = 56.7%5mC; mean post-exposure = 47.9%5mC; p-value = 0.009), whereas the mean methylation of HERV-w did not significantly differ. Individual exposure to inhalable particles and metals was estimated based on measures in all working areas and time spent by the study subjects in each area. In a regression model adjusted for age, body mass index and smoking, PM and metal components had a positive association with EBV-Wp methylation (i.e. PM10: β = 5.99, p-value < 0.038; nickel: β = 17.82, p-value = 0.02; arsenic: β = 13.59, p-value < 0.015). The difference observed comparing baseline and post-exposure samples may be suggestive of a rapid change in EBV methylation induced by air particles, while correlation between EBV methylation and PM/metal exposure may represent a more stable adaptive mechanism. Future studies investigating a larger panel of viral sequences could better elucidate

  18. Implementing targeted region capture sequencing for the clinical detection of Alagille syndrome: An efficient and cost‑effective method.

    PubMed

    Huang, Tianhong; Yang, Guilin; Dang, Xiao; Ao, Feijian; Li, Jiankang; He, Yizhou; Tang, Qiyuan; He, Qing

    2017-11-01

    Alagille syndrome (AGS) is a highly variable, autosomal dominant disease that affects multiple structures including the liver, heart, eyes, bones and face. Targeted region capture sequencing focuses on a panel of known pathogenic genes and provides a rapid, cost‑effective and accurate method for molecular diagnosis. In a Chinese family, this method was used on the proband and Sanger sequencing was applied to validate the candidate mutation. A de novo heterozygous mutation (c.3254_3255insT p.Leu1085PhefsX24) of the jagged 1 gene was identified as the potential disease‑causing gene mutation. In conclusion, the present study suggested that target region capture sequencing is an efficient, reliable and accurate approach for the clinical diagnosis of AGS. Furthermore, these results expand on the understanding of the pathogenesis of AGS.

  19. Effects of Early and Late Rest Intervals on Performance and Overnight Consolidation of a Keyboard Sequence

    ERIC Educational Resources Information Center

    Cash, Carla Davis

    2009-01-01

    Thirty-six nonmusicians practiced a five-element key-press sequence on a digital piano, repeating the sequence as quickly and accurately as possible during twelve 30-s practice blocks alternating with 30-s pauses. Twelve learners rested for 5 min between Blocks 3 and 4, another 12 learners rested for 5 min between Blocks 9 and 10, and the…

  20. A dental vision system for accurate 3D tooth modeling.

    PubMed

    Zhang, Li; Alemzadeh, K

    2006-01-01

    This paper describes an active vision system based reverse engineering approach to extract the three-dimensional (3D) geometric information from dental teeth and transfer this information into Computer-Aided Design/Computer-Aided Manufacture (CAD/CAM) systems to improve the accuracy of 3D teeth models and at the same time improve the quality of the construction units to help patient care. The vision system involves the development of a dental vision rig, edge detection, boundary tracing and fast & accurate 3D modeling from a sequence of sliced silhouettes of physical models. The rig is designed using engineering design methods such as a concept selection matrix and weighted objectives evaluation chart. Reconstruction results and accuracy evaluation are presented on digitizing different teeth models.

  1. BLAST and FASTA similarity searching for multiple sequence alignment.

    PubMed

    Pearson, William R

    2014-01-01

    BLAST, FASTA, and other similarity searching programs seek to identify homologous proteins and DNA sequences based on excess sequence similarity. If two sequences share much more similarity than expected by chance, the simplest explanation for the excess similarity is common ancestry-homology. The most effective similarity searches compare protein sequences, rather than DNA sequences, for sequences that encode proteins, and use expectation values, rather than percent identity, to infer homology. The BLAST and FASTA packages of sequence comparison programs provide programs for comparing protein and DNA sequences to protein databases (the most sensitive searches). Protein and translated-DNA comparisons to protein databases routinely allow evolutionary look back times from 1 to 2 billion years; DNA:DNA searches are 5-10-fold less sensitive. BLAST and FASTA can be run on popular web sites, but can also be downloaded and installed on local computers. With local installation, target databases can be customized for the sequence data being characterized. With today's very large protein databases, search sensitivity can also be improved by searching smaller comprehensive databases, for example, a complete protein set from an evolutionarily neighboring model organism. By default, BLAST and FASTA use scoring strategies target for distant evolutionary relationships; for comparisons involving short domains or queries, or searches that seek relatively close homologs (e.g. mouse-human), shallower scoring matrices will be more effective. Both BLAST and FASTA provide very accurate statistical estimates, which can be used to reliably identify protein sequences that diverged more than 2 billion years ago.

  2. Developing market class specific InDel markers from next generation sequence data in Phaseolus vulgaris L.

    PubMed

    Moghaddam, Samira Mafi; Song, Qijian; Mamidi, Sujan; Schmutz, Jeremy; Lee, Rian; Cregan, Perry; Osorno, Juan M; McClean, Phillip E

    2014-01-01

    Next generation sequence data provides valuable information and tools for genetic and genomic research and offers new insights useful for marker development. This data is useful for the design of accurate and user-friendly molecular tools. Common bean (Phaseolus vulgaris L.) is a diverse crop in which separate domestication events happened in each gene pool followed by race and market class diversification that has resulted in different morphological characteristics in each commercial market class. This has led to essentially independent breeding programs within each market class which in turn has resulted in limited within market class sequence variation. Sequence data from selected genotypes of five bean market classes (pinto, black, navy, and light and dark red kidney) were used to develop InDel-based markers specific to each market class. Design of the InDel markers was conducted through a combination of assembly, alignment and primer design software using 1.6× to 5.1× coverage of Illumina GAII sequence data for each of the selected genotypes. The procedure we developed for primer design is fast, accurate, less error prone, and higher throughput than when they are designed manually. All InDel markers are easy to run and score with no need for PCR optimization. A total of 2687 InDel markers distributed across the genome were developed. To highlight their usefulness, they were employed to construct a phylogenetic tree and a genetic map, showing that InDel markers are reliable, simple, and accurate.

  3. Developing market class specific InDel markers from next generation sequence data in Phaseolus vulgaris L.

    PubMed Central

    Moghaddam, Samira Mafi; Song, Qijian; Mamidi, Sujan; Schmutz, Jeremy; Lee, Rian; Cregan, Perry; Osorno, Juan M.; McClean, Phillip E.

    2013-01-01

    Next generation sequence data provides valuable information and tools for genetic and genomic research and offers new insights useful for marker development. This data is useful for the design of accurate and user-friendly molecular tools. Common bean (Phaseolus vulgaris L.) is a diverse crop in which separate domestication events happened in each gene pool followed by race and market class diversification that has resulted in different morphological characteristics in each commercial market class. This has led to essentially independent breeding programs within each market class which in turn has resulted in limited within market class sequence variation. Sequence data from selected genotypes of five bean market classes (pinto, black, navy, and light and dark red kidney) were used to develop InDel-based markers specific to each market class. Design of the InDel markers was conducted through a combination of assembly, alignment and primer design software using 1.6× to 5.1× coverage of Illumina GAII sequence data for each of the selected genotypes. The procedure we developed for primer design is fast, accurate, less error prone, and higher throughput than when they are designed manually. All InDel markers are easy to run and score with no need for PCR optimization. A total of 2687 InDel markers distributed across the genome were developed. To highlight their usefulness, they were employed to construct a phylogenetic tree and a genetic map, showing that InDel markers are reliable, simple, and accurate. PMID:24860578

  4. Validation of Genotyping-By-Sequencing Analysis in Populations of Tetraploid Alfalfa by 454 Sequencing

    PubMed Central

    Rocher, Solen; Jean, Martine; Castonguay, Yves; Belzile, François

    2015-01-01

    Genotyping-by-sequencing (GBS) is a relatively low-cost high throughput genotyping technology based on next generation sequencing and is applicable to orphan species with no reference genome. A combination of genome complexity reduction and multiplexing with DNA barcoding provides a simple and affordable way to resolve allelic variation between plant samples or populations. GBS was performed on ApeKI libraries using DNA from 48 genotypes each of two heterogeneous populations of tetraploid alfalfa (Medicago sativa spp. sativa): the synthetic cultivar Apica (ATF0) and a derived population (ATF5) obtained after five cycles of recurrent selection for superior tolerance to freezing (TF). Nearly 400 million reads were obtained from two lanes of an Illumina HiSeq 2000 sequencer and analyzed with the Universal Network-Enabled Analysis Kit (UNEAK) pipeline designed for species with no reference genome. Following the application of whole dataset-level filters, 11,694 single nucleotide polymorphism (SNP) loci were obtained. About 60% had a significant match on the Medicago truncatula syntenic genome. The accuracy of allelic ratios and genotype calls based on GBS data was directly assessed using 454 sequencing on a subset of SNP loci scored in eight plant samples. Sequencing depth in this study was not sufficient for accurate tetraploid allelic dosage, but reliable genotype calls based on diploid allelic dosage were obtained when using additional quality filtering. Principal Component Analysis of SNP loci in plant samples revealed that a small proportion (<5%) of the genetic variability assessed by GBS is able to differentiate ATF0 and ATF5. Our results confirm that analysis of GBS data using UNEAK is a reliable approach for genome-wide discovery of SNP loci in outcrossed polyploids. PMID:26115486

  5. Flow cytometry for enrichment and titration in massively parallel DNA sequencing

    PubMed Central

    Sandberg, Julia; Ståhl, Patrik L.; Ahmadian, Afshin; Bjursell, Magnus K.; Lundeberg, Joakim

    2009-01-01

    Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols. PMID:19304748

  6. Identifying novel sequence variants of RNA 3D motifs

    PubMed Central

    Zirbel, Craig L.; Roll, James; Sweeney, Blake A.; Petrov, Anton I.; Pirrung, Meg; Leontis, Neocles B.

    2015-01-01

    Predicting RNA 3D structure from sequence is a major challenge in biophysics. An important sub-goal is accurately identifying recurrent 3D motifs from RNA internal and hairpin loop sequences extracted from secondary structure (2D) diagrams. We have developed and validated new probabilistic models for 3D motif sequences based on hybrid Stochastic Context-Free Grammars and Markov Random Fields (SCFG/MRF). The SCFG/MRF models are constructed using atomic-resolution RNA 3D structures. To parameterize each model, we use all instances of each motif found in the RNA 3D Motif Atlas and annotations of pairwise nucleotide interactions generated by the FR3D software. Isostericity relations between non-Watson–Crick basepairs are used in scoring sequence variants. SCFG techniques model nested pairs and insertions, while MRF ideas handle crossing interactions and base triples. We use test sets of randomly-generated sequences to set acceptance and rejection thresholds for each motif group and thus control the false positive rate. Validation was carried out by comparing results for four motif groups to RMDetect. The software developed for sequence scoring (JAR3D) is structured to automatically incorporate new motifs as they accumulate in the RNA 3D Motif Atlas when new structures are solved and is available free for download. PMID:26130723

  7. The LAM-PCR Method to Sequence LV Integration Sites.

    PubMed

    Wang, Wei; Bartholomae, Cynthia C; Gabriel, Richard; Deichmann, Annette; Schmidt, Manfred

    2016-01-01

    Integrating viral gene transfer vectors are commonly used gene delivery tools in clinical gene therapy trials providing stable integration and continuous gene expression of the transgene in the treated host cell. However, integration of the reverse-transcribed vector DNA into the host genome is a potentially mutagenic event that may directly contribute to unwanted side effects. A comprehensive and accurate analysis of the integration site (IS) repertoire is indispensable to study clonality in transduced cells obtained from patients undergoing gene therapy and to identify potential in vivo selection of affected cell clones. To date, next-generation sequencing (NGS) of vector-genome junctions allows sophisticated studies on the integration repertoire in vitro and in vivo. We have explored the use of the Illumina MiSeq Personal Sequencer platform to sequence vector ISs amplified by non-restrictive linear amplification-mediated PCR (nrLAM-PCR) and LAM-PCR. MiSeq-based high-quality IS sequence retrieval is accomplished by the introduction of a double-barcode strategy that substantially minimizes the frequency of IS sequence collisions compared to the conventionally used single-barcode protocol. Here, we present an updated protocol of (nr)LAM-PCR for the analysis of lentiviral IS using a double-barcode system and followed by deep sequencing using the MiSeq device.

  8. Multiple echo multi-shot diffusion sequence.

    PubMed

    Chabert, Steren; Galindo, César; Tejos, Cristian; Uribe, Sergio A

    2014-04-01

    To measure both transversal relaxation time (T2 ) and diffusion coefficients within a single scan using a multi-shot approach. Both measurements have drawn interest in many applications, especially in skeletal muscle studies, which have short T2 values. Multiple echo single-shot schemes have been proposed to obtain those variables simultaneously within a single scan, resulting in a reduction of the scanning time. However, one problem with those approaches is the associated long echo read-out. Consequently, the minimum achievable echo time tends to be long, limiting the application of these sequences to tissues with relatively long T2 . To address this problem, we propose to extend the multi-echo sequences using a multi-shot approach, so that to allow shorter echo times. A multi-shot dual-echo EPI sequence with diffusion gradients and echo navigators was modified to include independent diffusion gradients in any of the two echoes. The multi-shot approach allows us to drastically reduce echo times. Results showed a good agreement for the T2 and mean diffusivity measurements with gold standard sequences in phantoms and in vivo data of calf muscles from healthy volunteers. A fast and accurate method is proposed to measure T2 and diffusion coefficients simultaneously, tested in vitro and in healthy volunteers. Copyright © 2013 Wiley Periodicals, Inc.

  9. DUK - A Fast and Efficient Kmer Based Sequence Matching Tool

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

    Li, Mingkun; Copeland, Alex; Han, James

    2011-03-21

    A new tool, DUK, is developed to perform matching task. Matching is to find whether a query sequence partially or totally matches given reference sequences or not. Matching is similar to alignment. Indeed many traditional analysis tasks like contaminant removal use alignment tools. But for matching, there is no need to know which bases of a query sequence matches which position of a reference sequence, it only need know whether there exists a match or not. This subtle difference can make matching task much faster than alignment. DUK is accurate, versatile, fast, and has efficient memory usage. It uses Kmermore » hashing method to index reference sequences and Poisson model to calculate p-value. DUK is carefully implemented in C++ in object oriented design. The resulted classes can also be used to develop other tools quickly. DUK have been widely used in JGI for a wide range of applications such as contaminant removal, organelle genome separation, and assembly refinement. Many real applications and simulated dataset demonstrate its power.« less

  10. Mechanisms controlling the complete accretionary beach state sequence

    NASA Astrophysics Data System (ADS)

    Dubarbier, Benjamin; Castelle, Bruno; Ruessink, Gerben; Marieu, Vincent

    2017-06-01

    Accretionary downstate beach sequence is a key element of observed nearshore morphological variability along sandy coasts. We present and analyze the first numerical simulation of such a sequence using a process-based morphodynamic model that solves the coupling between waves, depth-integrated currents, and sediment transport. The simulation evolves from an alongshore uniform barred beach (storm profile) to an almost featureless shore-welded terrace (summer profile) through the highly alongshore variable detached crescentic bar and transverse bar/rip system states. A global analysis of the full sequence allows determining the varying contributions of the different hydro-sedimentary processes. Sediment transport driven by orbital velocity skewness is critical to the overall onshore sandbar migration, while gravitational downslope sediment transport acts as a damping term inhibiting further channel growth enforced by rip flow circulation. Accurate morphological diffusivity and inclusion of orbital velocity skewness opens new perspectives in terms of morphodynamic modeling of real beaches.

  11. Accurate indel prediction using paired-end short reads

    PubMed Central

    2013-01-01

    Background One of the major open challenges in next generation sequencing (NGS) is the accurate identification of structural variants such as insertions and deletions (indels). Current methods for indel calling assign scores to different types of evidence or counter-evidence for the presence of an indel, such as the number of split read alignments spanning the boundaries of a deletion candidate or reads that map within a putative deletion. Candidates with a score above a manually defined threshold are then predicted to be true indels. As a consequence, structural variants detected in this manner contain many false positives. Results Here, we present a machine learning based method which is able to discover and distinguish true from false indel candidates in order to reduce the false positive rate. Our method identifies indel candidates using a discriminative classifier based on features of split read alignment profiles and trained on true and false indel candidates that were validated by Sanger sequencing. We demonstrate the usefulness of our method with paired-end Illumina reads from 80 genomes of the first phase of the 1001 Genomes Project ( http://www.1001genomes.org) in Arabidopsis thaliana. Conclusion In this work we show that indel classification is a necessary step to reduce the number of false positive candidates. We demonstrate that missing classification may lead to spurious biological interpretations. The software is available at: http://agkb.is.tuebingen.mpg.de/Forschung/SV-M/. PMID:23442375

  12. Fast alignment-free sequence comparison using spaced-word frequencies.

    PubMed

    Leimeister, Chris-Andre; Boden, Marcus; Horwege, Sebastian; Lindner, Sebastian; Morgenstern, Burkhard

    2014-07-15

    Alignment-free methods for sequence comparison are increasingly used for genome analysis and phylogeny reconstruction; they circumvent various difficulties of traditional alignment-based approaches. In particular, alignment-free methods are much faster than pairwise or multiple alignments. They are, however, less accurate than methods based on sequence alignment. Most alignment-free approaches work by comparing the word composition of sequences. A well-known problem with these methods is that neighbouring word matches are far from independent. To reduce the statistical dependency between adjacent word matches, we propose to use 'spaced words', defined by patterns of 'match' and 'don't care' positions, for alignment-free sequence comparison. We describe a fast implementation of this approach using recursive hashing and bit operations, and we show that further improvements can be achieved by using multiple patterns instead of single patterns. To evaluate our approach, we use spaced-word frequencies as a basis for fast phylogeny reconstruction. Using real-world and simulated sequence data, we demonstrate that our multiple-pattern approach produces better phylogenies than approaches relying on contiguous words. Our program is freely available at http://spaced.gobics.de/. © The Author 2014. Published by Oxford University Press.

  13. MetaPSICOV: combining coevolution methods for accurate prediction of contacts and long range hydrogen bonding in proteins.

    PubMed

    Jones, David T; Singh, Tanya; Kosciolek, Tomasz; Tetchner, Stuart

    2015-04-01

    Recent developments of statistical techniques to infer direct evolutionary couplings between residue pairs have rendered covariation-based contact prediction a viable means for accurate 3D modelling of proteins, with no information other than the sequence required. To extend the usefulness of contact prediction, we have designed a new meta-predictor (MetaPSICOV) which combines three distinct approaches for inferring covariation signals from multiple sequence alignments, considers a broad range of other sequence-derived features and, uniquely, a range of metrics which describe both the local and global quality of the input multiple sequence alignment. Finally, we use a two-stage predictor, where the second stage filters the output of the first stage. This two-stage predictor is additionally evaluated on its ability to accurately predict the long range network of hydrogen bonds, including correctly assigning the donor and acceptor residues. Using the original PSICOV benchmark set of 150 protein families, MetaPSICOV achieves a mean precision of 0.54 for top-L predicted long range contacts-around 60% higher than PSICOV, and around 40% better than CCMpred. In de novo protein structure prediction using FRAGFOLD, MetaPSICOV is able to improve the TM-scores of models by a median of 0.05 compared with PSICOV. Lastly, for predicting long range hydrogen bonding, MetaPSICOV-HB achieves a precision of 0.69 for the top-L/10 hydrogen bonds compared with just 0.26 for the baseline MetaPSICOV. MetaPSICOV is available as a freely available web server at http://bioinf.cs.ucl.ac.uk/MetaPSICOV. Raw data (predicted contact lists and 3D models) and source code can be downloaded from http://bioinf.cs.ucl.ac.uk/downloads/MetaPSICOV. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press.

  14. BONSAI Garden: Parallel knowledge discovery system for amino acid sequences

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

    Shoudai, T.; Miyano, S.; Shinohara, A.

    1995-12-31

    We have developed a machine discovery system BON-SAI which receives positive and negative examples as inputs and produces as a hypothesis a pair of a decision tree over regular patterns and an alphabet indexing. This system has succeeded in discovering reasonable knowledge on transmembrane domain sequences and signal peptide sequences by computer experiments. However, when several kinds of sequences axe mixed in the data, it does not seem reasonable for a single BONSAI system to find a hypothesis of a reasonably small size with high accuracy. For this purpose, we have designed a system BONSAI Garden, in which several BONSAI`smore » and a program called Gardener run over a network in parallel, to partition the data into some number of classes together with hypotheses explaining these classes accurately.« less

  15. Reducing assembly complexity of microbial genomes with single-molecule sequencing.

    PubMed

    Koren, Sergey; Harhay, Gregory P; Smith, Timothy P L; Bono, James L; Harhay, Dayna M; Mcvey, Scott D; Radune, Diana; Bergman, Nicholas H; Phillippy, Adam M

    2013-01-01

    The short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem. To measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads. Automated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

  16. Ab initio gene identification in metagenomic sequences

    PubMed Central

    Zhu, Wenhan; Lomsadze, Alexandre; Borodovsky, Mark

    2010-01-01

    We describe an algorithm for gene identification in DNA sequences derived from shotgun sequencing of microbial communities. Accurate ab initio gene prediction in a short nucleotide sequence of anonymous origin is hampered by uncertainty in model parameters. While several machine learning approaches could be proposed to bypass this difficulty, one effective method is to estimate parameters from dependencies, formed in evolution, between frequencies of oligonucleotides in protein-coding regions and genome nucleotide composition. Original version of the method was proposed in 1999 and has been used since for (i) reconstructing codon frequency vector needed for gene finding in viral genomes and (ii) initializing parameters of self-training gene finding algorithms. With advent of new prokaryotic genomes en masse it became possible to enhance the original approach by using direct polynomial and logistic approximations of oligonucleotide frequencies, as well as by separating models for bacteria and archaea. These advances have increased the accuracy of model reconstruction and, subsequently, gene prediction. We describe the refined method and assess its accuracy on known prokaryotic genomes split into short sequences. Also, we show that as a result of application of the new method, several thousands of new genes could be added to existing annotations of several human and mouse gut metagenomes. PMID:20403810

  17. The 2012 Emilia seismic sequence (Northern Italy): Imaging the thrust fault system by accurate aftershock location

    NASA Astrophysics Data System (ADS)

    Govoni, Aladino; Marchetti, Alessandro; De Gori, Pasquale; Di Bona, Massimo; Lucente, Francesco Pio; Improta, Luigi; Chiarabba, Claudio; Nardi, Anna; Margheriti, Lucia; Agostinetti, Nicola Piana; Di Giovambattista, Rita; Latorre, Diana; Anselmi, Mario; Ciaccio, Maria Grazia; Moretti, Milena; Castellano, Corrado; Piccinini, Davide

    2014-05-01

    Starting from late May 2012, the Emilia region (Northern Italy) was severely shaken by an intense seismic sequence, originated from a ML 5.9 earthquake on May 20th, at a hypocentral depth of 6.3 km, with thrust-type focal mechanism. In the following days, the seismic rate remained high, counting 50 ML ≥ 2.0 earthquakes a day, on average. Seismicity spreads along a 30 km east-west elongated area, in the Po river alluvial plain, in the nearby of the cities Ferrara and Modena. Nine days after the first shock, another destructive thrust-type earthquake (ML 5.8) hit the area to the west, causing further damage and fatalities. Aftershocks following this second destructive event extended along the same east-westerly trend for further 20 km to the west, thus illuminating an area of about 50 km in length, on the whole. After the first shock struck, on May 20th, a dense network of temporary seismic stations, in addition to the permanent ones, was deployed in the meizoseismal area, leading to a sensible improvement of the earthquake monitoring capability there. A combined dataset, including three-component seismic waveforms recorded by both permanent and temporary stations, has been analyzed in order to obtain an appropriate 1-D velocity model for earthquake location in the study area. Here we describe the main seismological characteristics of this seismic sequence and, relying on refined earthquakes location, we make inferences on the geometry of the thrust system responsible for the two strongest shocks.

  18. Correlation approach to identify coding regions in DNA sequences

    NASA Technical Reports Server (NTRS)

    Ossadnik, S. M.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Mantegna, R. N.; Peng, C. K.; Simons, M.; Stanley, H. E.

    1994-01-01

    Recently, it was observed that noncoding regions of DNA sequences possess long-range power-law correlations, whereas coding regions typically display only short-range correlations. We develop an algorithm based on this finding that enables investigators to perform a statistical analysis on long DNA sequences to locate possible coding regions. The algorithm is particularly successful in predicting the location of lengthy coding regions. For example, for the complete genome of yeast chromosome III (315,344 nucleotides), at least 82% of the predictions correspond to putative coding regions; the algorithm correctly identified all coding regions larger than 3000 nucleotides, 92% of coding regions between 2000 and 3000 nucleotides long, and 79% of coding regions between 1000 and 2000 nucleotides. The predictive ability of this new algorithm supports the claim that there is a fundamental difference in the correlation property between coding and noncoding sequences. This algorithm, which is not species-dependent, can be implemented with other techniques for rapidly and accurately locating relatively long coding regions in genomic sequences.

  19. The transition to increased automaticity during finger sequence learning in adult males who stutter.

    PubMed

    Smits-Bandstra, Sarah; De Nil, Luc; Rochon, Elizabeth

    2006-01-01

    The present study compared the automaticity levels of persons who stutter (PWS) and persons who do not stutter (PNS) on a practiced finger sequencing task under dual task conditions. Automaticity was defined as the amount of attention required for task performance. Twelve PWS and 12 control subjects practiced finger tapping sequences under single and then dual task conditions. Control subjects performed the sequencing task significantly faster and less variably under single versus dual task conditions while PWS' performance was consistently slow and variable (comparable to the dual task performance of control subjects) under both conditions. Control subjects were significantly more accurate on a colour recognition distracter task than PWS under dual task conditions. These results suggested that control subjects transitioned to quick, accurate and increasingly automatic performance on the sequencing task after practice, while PWS did not. Because most stuttering treatment programs for adults include practice and automatization of new motor speech skills, findings of this finger sequencing study and future studies of speech sequence learning may have important implications for how to maximize stuttering treatment effectiveness. As a result of this activity, the participant will be able to: (1) Define automaticity and explain the importance of dual task paradigms to investigate automaticity; (2) Relate the proposed relationship between motor learning and automaticity as stated by the authors; (3) Summarize the reviewed literature concerning the performance of PWS on dual tasks; and (4) Explain why the ability to transition to automaticity during motor learning may have important clinical implications for stuttering treatment effectiveness.

  20. Evolutionary profiles from the QR factorization of multiple sequence alignments

    PubMed Central

    Sethi, Anurag; O'Donoghue, Patrick; Luthey-Schulten, Zaida

    2005-01-01

    We present an algorithm to generate complete evolutionary profiles that represent the topology of the molecular phylogenetic tree of the homologous group. The method, based on the multidimensional QR factorization of numerically encoded multiple sequence alignments, removes redundancy from the alignments and orders the protein sequences by increasing linear dependence, resulting in the identification of a minimal basis set of sequences that spans the evolutionary space of the homologous group of proteins. We observe a general trend that these smaller, more evolutionarily balanced profiles have comparable and, in many cases, better performance in database searches than conventional profiles containing hundreds of sequences, constructed in an iterative and computationally intensive procedure. For more diverse families or superfamilies, with sequence identity <30%, structural alignments, based purely on the geometry of the protein structures, provide better alignments than pure sequence-based methods. Merging the structure and sequence information allows the construction of accurate profiles for distantly related groups. These structure-based profiles outperformed other sequence-based methods for finding distant homologs and were used to identify a putative class II cysteinyl-tRNA synthetase (CysRS) in several archaea that eluded previous annotation studies. Phylogenetic analysis showed the putative class II CysRSs to be a monophyletic group and homology modeling revealed a constellation of active site residues similar to that in the known class I CysRS. PMID:15741270

  1. Novel methodologies for spectral classification of exon and intron sequences

    NASA Astrophysics Data System (ADS)

    Kwan, Hon Keung; Kwan, Benjamin Y. M.; Kwan, Jennifer Y. Y.

    2012-12-01

    Digital processing of a nucleotide sequence requires it to be mapped to a numerical sequence in which the choice of nucleotide to numeric mapping affects how well its biological properties can be preserved and reflected from nucleotide domain to numerical domain. Digital spectral analysis of nucleotide sequences unfolds a period-3 power spectral value which is more prominent in an exon sequence as compared to that of an intron sequence. The success of a period-3 based exon and intron classification depends on the choice of a threshold value. The main purposes of this article are to introduce novel codes for 1-sequence numerical representations for spectral analysis and compare them to existing codes to determine appropriate representation, and to introduce novel thresholding methods for more accurate period-3 based exon and intron classification of an unknown sequence. The main findings of this study are summarized as follows: Among sixteen 1-sequence numerical representations, the K-Quaternary Code I offers an attractive performance. A windowed 1-sequence numerical representation (with window length of 9, 15, and 24 bases) offers a possible speed gain over non-windowed 4-sequence Voss representation which increases as sequence length increases. A winner threshold value (chosen from the best among two defined threshold values and one other threshold value) offers a top precision for classifying an unknown sequence of specified fixed lengths. An interpolated winner threshold value applicable to an unknown and arbitrary length sequence can be estimated from the winner threshold values of fixed length sequences with a comparable performance. In general, precision increases as sequence length increases. The study contributes an effective spectral analysis of nucleotide sequences to better reveal embedded properties, and has potential applications in improved genome annotation.

  2. Genome-wide DNA methylation analysis in jejunum of Sus scrofa with intrauterine growth restriction.

    PubMed

    Hu, Yue; Hu, Liang; Gong, Desheng; Lu, Hanlin; Xuan, Yue; Wang, Ru; Wu, De; Chen, Daiwen; Zhang, Keying; Gao, Fei; Che, Lianqiang

    2018-02-01

    Intrauterine growth restriction (IUGR) may elicit a series of postnatal body developmental and metabolic diseases due to their impaired growth and development in the mammalian embryo/fetus during pregnancy. In the present study, we hypothesized that IUGR may lead to abnormally regulated DNA methylation in the intestine, causing intestinal dysfunctions. We applied reduced representation bisulfite sequencing (RRBS) technology to study the jejunum tissues from four newborn IUGR piglets and their normal body weight (NBW) littermates. The results revealed extensively regional DNA methylation changes between IUGR/NBW pairs from different gilts, affecting dozens of genes. Hiseq-based bisulfite sequencing PCR (Hiseq-BSP) was used for validations of 19 genes with epigenetic abnormality, confirming three genes (AIFM1, MTMR1, and TWIST2) in extra samples. Furthermore, integrated analysis of these 19 genes with proteome data indicated that there were three main genes (BCAP31, IRAK1, and AIFM1) interacting with important immunity- or metabolism-related proteins, which could explain the potential intestinal dysfunctions of IUGR piglets. We conclude that IUGR can lead to disparate DNA methylation in the intestine and these changes may affect several important biological processes such as cell apoptosis, cell differentiation, and immunity, which provides more clues linking IUGR and its long-term complications.

  3. Compression-based distance (CBD): a simple, rapid, and accurate method for microbiota composition comparison

    PubMed Central

    2013-01-01

    Background Perturbations in intestinal microbiota composition have been associated with a variety of gastrointestinal tract-related diseases. The alleviation of symptoms has been achieved using treatments that alter the gastrointestinal tract microbiota toward that of healthy individuals. Identifying differences in microbiota composition through the use of 16S rRNA gene hypervariable tag sequencing has profound health implications. Current computational methods for comparing microbial communities are usually based on multiple alignments and phylogenetic inference, making them time consuming and requiring exceptional expertise and computational resources. As sequencing data rapidly grows in size, simpler analysis methods are needed to meet the growing computational burdens of microbiota comparisons. Thus, we have developed a simple, rapid, and accurate method, independent of multiple alignments and phylogenetic inference, to support microbiota comparisons. Results We create a metric, called compression-based distance (CBD) for quantifying the degree of similarity between microbial communities. CBD uses the repetitive nature of hypervariable tag datasets and well-established compression algorithms to approximate the total information shared between two datasets. Three published microbiota datasets were used as test cases for CBD as an applicable tool. Our study revealed that CBD recaptured 100% of the statistically significant conclusions reported in the previous studies, while achieving a decrease in computational time required when compared to similar tools without expert user intervention. Conclusion CBD provides a simple, rapid, and accurate method for assessing distances between gastrointestinal tract microbiota 16S hypervariable tag datasets. PMID:23617892

  4. Compression-based distance (CBD): a simple, rapid, and accurate method for microbiota composition comparison.

    PubMed

    Yang, Fang; Chia, Nicholas; White, Bryan A; Schook, Lawrence B

    2013-04-23

    Perturbations in intestinal microbiota composition have been associated with a variety of gastrointestinal tract-related diseases. The alleviation of symptoms has been achieved using treatments that alter the gastrointestinal tract microbiota toward that of healthy individuals. Identifying differences in microbiota composition through the use of 16S rRNA gene hypervariable tag sequencing has profound health implications. Current computational methods for comparing microbial communities are usually based on multiple alignments and phylogenetic inference, making them time consuming and requiring exceptional expertise and computational resources. As sequencing data rapidly grows in size, simpler analysis methods are needed to meet the growing computational burdens of microbiota comparisons. Thus, we have developed a simple, rapid, and accurate method, independent of multiple alignments and phylogenetic inference, to support microbiota comparisons. We create a metric, called compression-based distance (CBD) for quantifying the degree of similarity between microbial communities. CBD uses the repetitive nature of hypervariable tag datasets and well-established compression algorithms to approximate the total information shared between two datasets. Three published microbiota datasets were used as test cases for CBD as an applicable tool. Our study revealed that CBD recaptured 100% of the statistically significant conclusions reported in the previous studies, while achieving a decrease in computational time required when compared to similar tools without expert user intervention. CBD provides a simple, rapid, and accurate method for assessing distances between gastrointestinal tract microbiota 16S hypervariable tag datasets.

  5. Flexible taxonomic assignment of ambiguous sequencing reads

    PubMed Central

    2011-01-01

    Background To characterize the diversity of bacterial populations in metagenomic studies, sequencing reads need to be accurately assigned to taxonomic units in a given reference taxonomy. Reads that cannot be reliably assigned to a unique leaf in the taxonomy (ambiguous reads) are typically assigned to the lowest common ancestor of the set of species that match it. This introduces a potentially severe error in the estimation of bacteria present in the sample due to false positives, since all species in the subtree rooted at the ancestor are implicitly assigned to the read even though many of them may not match it. Results We present a method that maps each read to a node in the taxonomy that minimizes a penalty score while balancing the relevance of precision and recall in the assignment through a parameter q. This mapping can be obtained in time linear in the number of matching sequences, because LCA queries to the reference taxonomy take constant time. When applied to six different metagenomic datasets, our algorithm produces different taxonomic distributions depending on whether coverage or precision is maximized. Including information on the quality of the reads reduces the number of unassigned reads but increases the number of ambiguous reads, stressing the relevance of our method. Finally, two measures of performance are described and results with a set of artificially generated datasets are discussed. Conclusions The assignment strategy of sequencing reads introduced in this paper is a versatile and a quick method to study bacterial communities. The bacterial composition of the analyzed samples can vary significantly depending on how ambiguous reads are assigned depending on the value of the q parameter. Validation of our results in an artificial dataset confirm that a combination of values of q produces the most accurate results. PMID:21211059

  6. CSReport: A New Computational Tool Designed for Automatic Analysis of Class Switch Recombination Junctions Sequenced by High-Throughput Sequencing.

    PubMed

    Boyer, François; Boutouil, Hend; Dalloul, Iman; Dalloul, Zeinab; Cook-Moreau, Jeanne; Aldigier, Jean-Claude; Carrion, Claire; Herve, Bastien; Scaon, Erwan; Cogné, Michel; Péron, Sophie

    2017-05-15

    B cells ensure humoral immune responses due to the production of Ag-specific memory B cells and Ab-secreting plasma cells. In secondary lymphoid organs, Ag-driven B cell activation induces terminal maturation and Ig isotype class switch (class switch recombination [CSR]). CSR creates a virtually unique IgH locus in every B cell clone by intrachromosomal recombination between two switch (S) regions upstream of each C region gene. Amount and structural features of CSR junctions reveal valuable information about the CSR mechanism, and analysis of CSR junctions is useful in basic and clinical research studies of B cell functions. To provide an automated tool able to analyze large data sets of CSR junction sequences produced by high-throughput sequencing (HTS), we designed CSReport, a software program dedicated to support analysis of CSR recombination junctions sequenced with a HTS-based protocol (Ion Torrent technology). CSReport was assessed using simulated data sets of CSR junctions and then used for analysis of Sμ-Sα and Sμ-Sγ1 junctions from CH12F3 cells and primary murine B cells, respectively. CSReport identifies junction segment breakpoints on reference sequences and junction structure (blunt-ended junctions or junctions with insertions or microhomology). Besides the ability to analyze unprecedentedly large libraries of junction sequences, CSReport will provide a unified framework for CSR junction studies. Our results show that CSReport is an accurate tool for analysis of sequences from our HTS-based protocol for CSR junctions, thereby facilitating and accelerating their study. Copyright © 2017 by The American Association of Immunologists, Inc.

  7. Accurate prediction of hot spot residues through physicochemical characteristics of amino acid sequences.

    PubMed

    Chen, Peng; Li, Jinyan; Wong, Limsoon; Kuwahara, Hiroyuki; Huang, Jianhua Z; Gao, Xin

    2013-08-01

    Hot spot residues of proteins are fundamental interface residues that help proteins perform their functions. Detecting hot spots by experimental methods is costly and time-consuming. Sequential and structural information has been widely used in the computational prediction of hot spots. However, structural information is not always available. In this article, we investigated the problem of identifying hot spots using only physicochemical characteristics extracted from amino acid sequences. We first extracted 132 relatively independent physicochemical features from a set of the 544 properties in AAindex1, an amino acid index database. Each feature was utilized to train a classification model with a novel encoding schema for hot spot prediction by the IBk algorithm, an extension of the K-nearest neighbor algorithm. The combinations of the individual classifiers were explored and the classifiers that appeared frequently in the top performing combinations were selected. The hot spot predictor was built based on an ensemble of these classifiers and to work in a voting manner. Experimental results demonstrated that our method effectively exploited the feature space and allowed flexible weights of features for different queries. On the commonly used hot spot benchmark sets, our method significantly outperformed other machine learning algorithms and state-of-the-art hot spot predictors. The program is available at http://sfb.kaust.edu.sa/pages/software.aspx. Copyright © 2013 Wiley Periodicals, Inc.

  8. A multi-objective optimization approach accurately resolves protein domain architectures

    PubMed Central

    Bernardes, J.S.; Vieira, F.R.J.; Zaverucha, G.; Carbone, A.

    2016-01-01

    Motivation: Given a protein sequence and a number of potential domains matching it, what are the domain content and the most likely domain architecture for the sequence? This problem is of fundamental importance in protein annotation, constituting one of the main steps of all predictive annotation strategies. On the other hand, when potential domains are several and in conflict because of overlapping domain boundaries, finding a solution for the problem might become difficult. An accurate prediction of the domain architecture of a multi-domain protein provides important information for function prediction, comparative genomics and molecular evolution. Results: We developed DAMA (Domain Annotation by a Multi-objective Approach), a novel approach that identifies architectures through a multi-objective optimization algorithm combining scores of domain matches, previously observed multi-domain co-occurrence and domain overlapping. DAMA has been validated on a known benchmark dataset based on CATH structural domain assignments and on the set of Plasmodium falciparum proteins. When compared with existing tools on both datasets, it outperforms all of them. Availability and implementation: DAMA software is implemented in C++ and the source code can be found at http://www.lcqb.upmc.fr/DAMA. Contact: juliana.silva_bernardes@upmc.fr or alessandra.carbone@lip6.fr Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26458889

  9. A pooling-based approach to mapping genetic variants associated with DNA methylation

    PubMed Central

    Kaplow, Irene M.; MacIsaac, Julia L.; Mah, Sarah M.; McEwen, Lisa M.; Kobor, Michael S.; Fraser, Hunter B.

    2015-01-01

    DNA methylation is an epigenetic modification that plays a key role in gene regulation. Previous studies have investigated its genetic basis by mapping genetic variants that are associated with DNA methylation at specific sites, but these have been limited to microarrays that cover <2% of the genome and cannot account for allele-specific methylation (ASM). Other studies have performed whole-genome bisulfite sequencing on a few individuals, but these lack statistical power to identify variants associated with DNA methylation. We present a novel approach in which bisulfite-treated DNA from many individuals is sequenced together in a single pool, resulting in a truly genome-wide map of DNA methylation. Compared to methods that do not account for ASM, our approach increases statistical power to detect associations while sharply reducing cost, effort, and experimental variability. As a proof of concept, we generated deep sequencing data from a pool of 60 human cell lines; we evaluated almost twice as many CpGs as the largest microarray studies and identified more than 2000 genetic variants associated with DNA methylation. We found that these variants are highly enriched for associations with chromatin accessibility and CTCF binding but are less likely to be associated with traits indirectly linked to DNA, such as gene expression and disease phenotypes. In summary, our approach allows genome-wide mapping of genetic variants associated with DNA methylation in any tissue of any species, without the need for individual-level genotype or methylation data. PMID:25910490

  10. Allele-specific copy-number discovery from whole-genome and whole-exome sequencing.

    PubMed

    Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J; Szatkiewicz, Jin P

    2015-08-18

    Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. QuickProbs—A Fast Multiple Sequence Alignment Algorithm Designed for Graphics Processors

    PubMed Central

    Gudyś, Adam; Deorowicz, Sebastian

    2014-01-01

    Multiple sequence alignment is a crucial task in a number of biological analyses like secondary structure prediction, domain searching, phylogeny, etc. MSAProbs is currently the most accurate alignment algorithm, but its effectiveness is obtained at the expense of computational time. In the paper we present QuickProbs, the variant of MSAProbs customised for graphics processors. We selected the two most time consuming stages of MSAProbs to be redesigned for GPU execution: the posterior matrices calculation and the consistency transformation. Experiments on three popular benchmarks (BAliBASE, PREFAB, OXBench-X) on quad-core PC equipped with high-end graphics card show QuickProbs to be 5.7 to 9.7 times faster than original CPU-parallel MSAProbs. Additional tests performed on several protein families from Pfam database give overall speed-up of 6.7. Compared to other algorithms like MAFFT, MUSCLE, or ClustalW, QuickProbs proved to be much more accurate at similar speed. Additionally we introduce a tuned variant of QuickProbs which is significantly more accurate on sets of distantly related sequences than MSAProbs without exceeding its computation time. The GPU part of QuickProbs was implemented in OpenCL, thus the package is suitable for graphics processors produced by all major vendors. PMID:24586435

  12. Towards accurate localization: long- and short-term correlation filters for tracking

    NASA Astrophysics Data System (ADS)

    Li, Minglangjun; Tian, Chunna

    2018-04-01

    Visual tracking is a challenging problem, especially using a single model. In this paper, we propose a discriminative correlation filter (DCF) based tracking approach that exploits both the long-term and short-term information of the target, named LSTDCF, to improve the tracking performance. In addition to a long-term filter learned through the whole sequence, a short-term filter is trained using only features extracted from most recent frames. The long-term filter tends to capture more semantics of the target as more frames are used for training. However, since the target may undergo large appearance changes, features extracted around the target in non-recent frames prevent the long-term filter from locating the target in the current frame accurately. In contrast, the short-term filter learns more spatial details of the target from recent frames but gets over-fitting easily. Thus the short-term filter is less robust to handle cluttered background and prone to drift. We take the advantage of both filters and fuse their response maps to make the final estimation. We evaluate our approach on a widely-used benchmark with 100 image sequences and achieve state-of-the-art results.

  13. Accurate measurement of heteronuclear dipolar couplings by phase-alternating R-symmetry (PARS) sequences in magic angle spinning NMR spectroscopy

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

    Hou, Guangjin, E-mail: hou@udel.edu, E-mail: tpolenov@udel.edu; Lu, Xingyu, E-mail: luxingyu@udel.edu, E-mail: lexvega@comcast.net; Vega, Alexander J., E-mail: luxingyu@udel.edu, E-mail: lexvega@comcast.net

    2014-09-14

    We report a Phase-Alternating R-Symmetry (PARS) dipolar recoupling scheme for accurate measurement of heteronuclear {sup 1}H-X (X = {sup 13}C, {sup 15}N, {sup 31}P, etc.) dipolar couplings in MAS NMR experiments. It is an improvement of conventional C- and R-symmetry type DIPSHIFT experiments where, in addition to the dipolar interaction, the {sup 1}H CSA interaction persists and thereby introduces considerable errors in the dipolar measurements. In PARS, phase-shifted RN symmetry pulse blocks applied on the {sup 1}H spins combined with π pulses applied on the X spins at the end of each RN block efficiently suppress the effect from {supmore » 1}H chemical shift anisotropy, while keeping the {sup 1}H-X dipolar couplings intact. Another advantage over conventional DIPSHIFT experiments, which require the signal to be detected in the form of a reduced-intensity Hahn echo, is that the series of π pulses refocuses the X chemical shift and avoids the necessity of echo formation. PARS permits determination of accurate dipolar couplings in a single experiment; it is suitable for a wide range of MAS conditions including both slow and fast MAS frequencies; and it assures dipolar truncation from the remote protons. The performance of PARS is tested on two model systems, [{sup 15}N]-N-acetyl-valine and [U-{sup 13}C,{sup 15}N]-N-formyl-Met-Leu-Phe tripeptide. The application of PARS for site-resolved measurement of accurate {sup 1}H-{sup 15}N dipolar couplings in the context of 3D experiments is presented on U-{sup 13}C,{sup 15}N-enriched dynein light chain protein LC8.« less

  14. Seismic sequences in the Sombrero Seismic Zone

    NASA Astrophysics Data System (ADS)

    Pulliam, J.; Huerfano, V. A.; ten Brink, U.; von Hillebrandt, C.

    2007-05-01

    The northeastern Caribbean, in the vicinity of Puerto Rico and the Virgin Islands, has a long and well-documented history of devastating earthquakes and tsunamis, including major events in 1670, 1787, 1867, 1916, 1918, and 1943. Recently, seismicity has been concentrated to the north and west of the British Virgin Islands, in the region referred to as the Sombrero Seismic Zone by the Puerto Rico Seismic Network (PRSN). In the combined seismicity catalog maintained by the PRSN, several hundred small to moderate magnitude events can be found in this region prior to 2006. However, beginning in 2006 and continuing to the present, the rate of seismicity in the Sombrero suddenly increased, and a new locus of activity developed to the east of the previous location. Accurate estimates of seismic hazard, and the tsunamigenic potential of seismic events, depend on an accurate and comprehensive understanding of how strain is being accommodated in this corner region. Are faults locked and accumulating strain for release in a major event? Or is strain being released via slip over a diffuse system of faults? A careful analysis of seismicity patterns in the Sombrero region has the potential to both identify faults and modes of failure, provided the aggregation scheme is tuned to properly identify related events. To this end, we experimented with a scheme to identify seismic sequences based on physical and temporal proximity, under the assumptions that (a) events occur on related fault systems as stress is refocused by immediately previous events and (b) such 'stress waves' die out with time, so that two events that occur on the same system within a relatively short time window can be said to have a similar 'trigger' in ways that two nearby events that occurred years apart cannot. Patterns that emerge from the identification, temporal sequence, and refined locations of such sequences of events carry information about stress accommodation that is obscured by large clouds of

  15. Pre-main Sequence Evolution and the Hydrogen-Burning Minimum Mass

    NASA Astrophysics Data System (ADS)

    Nakano, Takenori

    There is a lower limit to the mass of the main-sequence stars (the hydrogen-burning minimum mass) below which the stars cannot replenish the energy lost from their surfaces with the energy released by the hydrogen burning in their cores. This is caused by the electron degeneracy in the stars which suppresses the increase of the central temperature with contraction. To find out the lower limit we need the accurate knowledge of the pre-main sequence evolution of very low-mass stars in which the effect of electron degeneracy is important. We review how Hayashi and Nakano (1963) carried out the first determination of this limit.

  16. High quality de novo sequencing and assembly of the Saccharomyces arboricolus genome

    PubMed Central

    2013-01-01

    Background Comparative genomics is a formidable tool to identify functional elements throughout a genome. In the past ten years, studies in the budding yeast Saccharomyces cerevisiae and a set of closely related species have been instrumental in showing the benefit of analyzing patterns of sequence conservation. Increasing the number of closely related genome sequences makes the comparative genomics approach more powerful and accurate. Results Here, we report the genome sequence and analysis of Saccharomyces arboricolus, a yeast species recently isolated in China, that is closely related to S. cerevisiae. We obtained high quality de novo sequence and assemblies using a combination of next generation sequencing technologies, established the phylogenetic position of this species and considered its phenotypic profile under multiple environmental conditions in the light of its gene content and phylogeny. Conclusions We suggest that the genome of S. arboricolus will be useful in future comparative genomics analysis of the Saccharomyces sensu stricto yeasts. PMID:23368932

  17. Complete mitochondrial genome sequence of northeastern sika deer (Cervus nippon hortulorum).

    PubMed

    Shao, Yuanchen; Zha, Daiming; Xing, Xiumei; Su, Weilin; Liu, Huamiao; Zhang, Ranran

    2016-01-01

    The complete mitochondrial genome of the northeastern sika deer, Cervus nippon hortulorum, was determined by accurate polymerase chain reaction. The entire genome is 16,434 bp in length and contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and 1 control region, all of which are arranged in a typical vertebrate manner. The overall base composition of the northeastern sika deer's mitochondrial genome is 33.3% of A, 24.5% of C, 28.7% of T and 13.5% of G. A termination associated sequence and several conserved central sequence block domains were discovered within the control region.

  18. An accurate and efficient experimental approach for characterization of the complex oral microbiota.

    PubMed

    Zheng, Wei; Tsompana, Maria; Ruscitto, Angela; Sharma, Ashu; Genco, Robert; Sun, Yijun; Buck, Michael J

    2015-10-05

    Currently, taxonomic interrogation of microbiota is based on amplification of 16S rRNA gene sequences in clinical and scientific settings. Accurate evaluation of the microbiota depends heavily on the primers used, and genus/species resolution bias can arise with amplification of non-representative genomic regions. The latest Illumina MiSeq sequencing chemistry has extended the read length to 300 bp, enabling deep profiling of large number of samples in a single paired-end reaction at a fraction of the cost. An increasingly large number of researchers have adopted this technology for various microbiome studies targeting the 16S rRNA V3-V4 hypervariable region. To expand the applicability of this powerful platform for further descriptive and functional microbiome studies, we standardized and tested an efficient, reliable, and straightforward workflow for the amplification, library construction, and sequencing of the 16S V1-V3 hypervariable region using the new 2 × 300 MiSeq platform. Our analysis involved 11 subgingival plaque samples from diabetic and non-diabetic human subjects suffering from periodontitis. The efficiency and reliability of our experimental protocol was compared to 16S V3-V4 sequencing data from the same samples. Comparisons were based on measures of observed taxonomic richness and species evenness, along with Procrustes analyses using beta(β)-diversity distance metrics. As an experimental control, we also analyzed a total of eight technical replicates for the V1-V3 and V3-V4 regions from a synthetic community with known bacterial species operon counts. We show that our experimental protocol accurately measures true bacterial community composition. Procrustes analyses based on unweighted UniFrac β-diversity metrics depicted significant correlation between oral bacterial composition for the V1-V3 and V3-V4 regions. However, measures of phylotype richness were higher for the V1-V3 region, suggesting that V1-V3 offers a deeper assessment of

  19. Nonspatial Sequence Coding in CA1 Neurons

    PubMed Central

    Allen, Timothy A.; Salz, Daniel M.; McKenzie, Sam

    2016-01-01

    The hippocampus is critical to the memory for sequences of events, a defining feature of episodic memory. However, the fundamental neuronal mechanisms underlying this capacity remain elusive. While considerable research indicates hippocampal neurons can represent sequences of locations, direct evidence of coding for the memory of sequential relationships among nonspatial events remains lacking. To address this important issue, we recorded neural activity in CA1 as rats performed a hippocampus-dependent sequence-memory task. Briefly, the task involves the presentation of repeated sequences of odors at a single port and requires rats to identify each item as “in sequence” or “out of sequence”. We report that, while the animals' location and behavior remained constant, hippocampal activity differed depending on the temporal context of items—in this case, whether they were presented in or out of sequence. Some neurons showed this effect across items or sequence positions (general sequence cells), while others exhibited selectivity for specific conjunctions of item and sequence position information (conjunctive sequence cells) or for specific probe types (probe-specific sequence cells). We also found that the temporal context of individual trials could be accurately decoded from the activity of neuronal ensembles, that sequence coding at the single-cell and ensemble level was linked to sequence memory performance, and that slow-gamma oscillations (20–40 Hz) were more strongly modulated by temporal context and performance than theta oscillations (4–12 Hz). These findings provide compelling evidence that sequence coding extends beyond the domain of spatial trajectories and is thus a fundamental function of the hippocampus. SIGNIFICANCE STATEMENT The ability to remember the order of life events depends on the hippocampus, but the underlying neural mechanisms remain poorly understood. Here we addressed this issue by recording neural activity in hippocampal

  20. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing

    PubMed Central

    Hykin, Sarah M.; Bi, Ke; McGuire, Jimmy A.

    2015-01-01

    For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens—particularly for use in phylogenetic analyses—has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for

  1. Fixing Formalin: A Method to Recover Genomic-Scale DNA Sequence Data from Formalin-Fixed Museum Specimens Using High-Throughput Sequencing.

    PubMed

    Hykin, Sarah M; Bi, Ke; McGuire, Jimmy A

    2015-01-01

    For 150 years or more, specimens were routinely collected and deposited in natural history collections without preserving fresh tissue samples for genetic analysis. In the case of most herpetological specimens (i.e. amphibians and reptiles), attempts to extract and sequence DNA from formalin-fixed, ethanol-preserved specimens-particularly for use in phylogenetic analyses-has been laborious and largely ineffective due to the highly fragmented nature of the DNA. As a result, tens of thousands of specimens in herpetological collections have not been available for sequence-based phylogenetic studies. Massively parallel High-Throughput Sequencing methods and the associated bioinformatics, however, are particularly suited to recovering meaningful genetic markers from severely degraded/fragmented DNA sequences such as DNA damaged by formalin-fixation. In this study, we compared previously published DNA extraction methods on three tissue types subsampled from formalin-fixed specimens of Anolis carolinensis, followed by sequencing. Sufficient quality DNA was recovered from liver tissue, making this technique minimally destructive to museum specimens. Sequencing was only successful for the more recently collected specimen (collected ~30 ybp). We suspect this could be due either to the conditions of preservation and/or the amount of tissue used for extraction purposes. For the successfully sequenced sample, we found a high rate of base misincorporation. After rigorous trimming, we successfully mapped 27.93% of the cleaned reads to the reference genome, were able to reconstruct the complete mitochondrial genome, and recovered an accurate phylogenetic placement for our specimen. We conclude that the amount of DNA available, which can vary depending on specimen age and preservation conditions, will determine if sequencing will be successful. The technique described here will greatly improve the value of museum collections by making many formalin-fixed specimens available for

  2. Thrombus segmentation by texture dynamics from microscopic image sequences

    NASA Astrophysics Data System (ADS)

    Brieu, Nicolas; Serbanovic-Canic, Jovana; Cvejic, Ana; Stemple, Derek; Ouwehand, Willem; Navab, Nassir; Groher, Martin

    2010-03-01

    The genetic factors of thrombosis are commonly explored by microscopically imaging the coagulation of blood cells induced by injuring a vessel of mice or of zebrafish mutants. The latter species is particularly interesting since skin transparency permits to non-invasively acquire microscopic images of the scene with a CCD camera and to estimate the parameters characterizing the thrombus development. These parameters are currently determined by manual outlining, which is both error prone and extremely time consuming. Even though a technique for automatic thrombus extraction would be highly valuable for gene analysts, little work can be found, which is mainly due to very low image contrast and spurious structures. In this work, we propose to semi-automatically segment the thrombus over time from microscopic image sequences of wild-type zebrafish larvae. To compensate the lack of valuable spatial information, our main idea consists of exploiting the temporal information by modeling the variations of the pixel intensities over successive temporal windows with a linear Markov-based dynamic texture formalization. We then derive an image from the estimated model parameters, which represents the probability of a pixel to belong to the thrombus. We employ this probability image to accurately estimate the thrombus position via an active contour segmentation incorporating also prior and spatial information of the underlying intensity images. The performance of our approach is tested on three microscopic image sequences. We show that the thrombus is accurately tracked over time in each sequence if the respective parameters controlling prior influence and contour stiffness are correctly chosen.

  3. 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

  4. A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION™ nanopore sequencer.

    PubMed

    Imai, Kazuo; Tarumoto, Norihito; Misawa, Kazuhisa; Runtuwene, Lucky Ronald; Sakai, Jun; Hayashida, Kyoko; Eshita, Yuki; Maeda, Ryuichiro; Tuda, Josef; Murakami, Takashi; Maesaki, Shigefumi; Suzuki, Yutaka; Yamagishi, Junya; Maeda, Takuya

    2017-09-13

    A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer. We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species. Our LAMP method allowed amplification of all targeted 18S-rRNA genes of the reference plasmids with detection limits of 10-100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR. Our diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

  5. Aftershock occurrence rate decay for individual sequences and catalogs

    NASA Astrophysics Data System (ADS)

    Nyffenegger, Paul A.

    One of the earliest observations of the Earth's seismicity is that the rate of aftershock occurrence decays with time according to a power law commonly known as modified Omori-law (MOL) decay. However, the physical reasons for aftershock occurrence and the empirical decay in rate remain unclear despite numerous models that yield similar rate decay behavior. Key problems in relating the observed empirical relationship to the physical conditions of the mainshock and fault are the lack of studies including small magnitude mainshocks and the lack of uniformity between studies. We use simulated aftershock sequences to investigate the factors which influence the maximum likelihood (ML) estimate of the Omori-law p value, the parameter describing aftershock occurrence rate decay, for both individual aftershock sequences and "stacked" or superposed sequences. Generally the ML estimate of p is accurate, but since the ML estimated uncertainty is unaffected by whether the sequence resembles an MOL model, a goodness-of-fit test such as the Anderson-Darling statistic is necessary. While stacking aftershock sequences permits the study of entire catalogs and sequences with small aftershock populations, stacking introduces artifacts. The p value for stacked sequences is approximately equal to the mean of the individual sequence p values. We apply single-link cluster analysis to identify all aftershock sequences from eleven regional seismicity catalogs. We observe two new mathematically predictable empirical relationships for the distribution of aftershock sequence populations. The average properties of aftershock sequences are not correlated with tectonic environment, but aftershock populations and p values do show a depth dependence. The p values show great variability with time, and large values or changes in p sometimes precedes major earthquakes. Studies of teleseismic earthquake catalogs over the last twenty years have led seismologists to question seismicity models and

  6. The sequence of sequencers: The history of sequencing DNA

    PubMed Central

    Heather, James M.; Chain, Benjamin

    2016-01-01

    Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way. PMID:26554401

  7. Accurate Classification of RNA Structures Using Topological Fingerprints

    PubMed Central

    Li, Kejie; Gribskov, Michael

    2016-01-01

    While RNAs are well known to possess complex structures, functionally similar RNAs often have little sequence similarity. While the exact size and spacing of base-paired regions vary, functionally similar RNAs have pronounced similarity in the arrangement, or topology, of base-paired stems. Furthermore, predicted RNA structures often lack pseudoknots (a crucial aspect of biological activity), and are only partially correct, or incomplete. A topological approach addresses all of these difficulties. In this work we describe each RNA structure as a graph that can be converted to a topological spectrum (RNA fingerprint). The set of subgraphs in an RNA structure, its RNA fingerprint, can be compared with the fingerprints of other RNA structures to identify and correctly classify functionally related RNAs. Topologically similar RNAs can be identified even when a large fraction, up to 30%, of the stems are omitted, indicating that highly accurate structures are not necessary. We investigate the performance of the RNA fingerprint approach on a set of eight highly curated RNA families, with diverse sizes and functions, containing pseudoknots, and with little sequence similarity–an especially difficult test set. In spite of the difficult test set, the RNA fingerprint approach is very successful (ROC AUC > 0.95). Due to the inclusion of pseudoknots, the RNA fingerprint approach both covers a wider range of possible structures than methods based only on secondary structure, and its tolerance for incomplete structures suggests that it can be applied even to predicted structures. Source code is freely available at https://github.rcac.purdue.edu/mgribsko/XIOS_RNA_fingerprint. PMID:27755571

  8. Accurate De Novo Prediction of Protein Contact Map by Ultra-Deep Learning Model.

    PubMed

    Wang, Sheng; Sun, Siqi; Li, Zhen; Zhang, Renyu; Xu, Jinbo

    2017-01-01

    Protein contacts contain key information for the understanding of protein structure and function and thus, contact prediction from sequence is an important problem. Recently exciting progress has been made on this problem, but the predicted contacts for proteins without many sequence homologs is still of low quality and not very useful for de novo structure prediction. This paper presents a new deep learning method that predicts contacts by integrating both evolutionary coupling (EC) and sequence conservation information through an ultra-deep neural network formed by two deep residual neural networks. The first residual network conducts a series of 1-dimensional convolutional transformation of sequential features; the second residual network conducts a series of 2-dimensional convolutional transformation of pairwise information including output of the first residual network, EC information and pairwise potential. By using very deep residual networks, we can accurately model contact occurrence patterns and complex sequence-structure relationship and thus, obtain higher-quality contact prediction regardless of how many sequence homologs are available for proteins in question. Our method greatly outperforms existing methods and leads to much more accurate contact-assisted folding. Tested on 105 CASP11 targets, 76 past CAMEO hard targets, and 398 membrane proteins, the average top L long-range prediction accuracy obtained by our method, one representative EC method CCMpred and the CASP11 winner MetaPSICOV is 0.47, 0.21 and 0.30, respectively; the average top L/10 long-range accuracy of our method, CCMpred and MetaPSICOV is 0.77, 0.47 and 0.59, respectively. Ab initio folding using our predicted contacts as restraints but without any force fields can yield correct folds (i.e., TMscore>0.6) for 203 of the 579 test proteins, while that using MetaPSICOV- and CCMpred-predicted contacts can do so for only 79 and 62 of them, respectively. Our contact-assisted models also have

  9. Accurate De Novo Prediction of Protein Contact Map by Ultra-Deep Learning Model

    PubMed Central

    Li, Zhen; Zhang, Renyu

    2017-01-01

    Motivation Protein contacts contain key information for the understanding of protein structure and function and thus, contact prediction from sequence is an important problem. Recently exciting progress has been made on this problem, but the predicted contacts for proteins without many sequence homologs is still of low quality and not very useful for de novo structure prediction. Method This paper presents a new deep learning method that predicts contacts by integrating both evolutionary coupling (EC) and sequence conservation information through an ultra-deep neural network formed by two deep residual neural networks. The first residual network conducts a series of 1-dimensional convolutional transformation of sequential features; the second residual network conducts a series of 2-dimensional convolutional transformation of pairwise information including output of the first residual network, EC information and pairwise potential. By using very deep residual networks, we can accurately model contact occurrence patterns and complex sequence-structure relationship and thus, obtain higher-quality contact prediction regardless of how many sequence homologs are available for proteins in question. Results Our method greatly outperforms existing methods and leads to much more accurate contact-assisted folding. Tested on 105 CASP11 targets, 76 past CAMEO hard targets, and 398 membrane proteins, the average top L long-range prediction accuracy obtained by our method, one representative EC method CCMpred and the CASP11 winner MetaPSICOV is 0.47, 0.21 and 0.30, respectively; the average top L/10 long-range accuracy of our method, CCMpred and MetaPSICOV is 0.77, 0.47 and 0.59, respectively. Ab initio folding using our predicted contacts as restraints but without any force fields can yield correct folds (i.e., TMscore>0.6) for 203 of the 579 test proteins, while that using MetaPSICOV- and CCMpred-predicted contacts can do so for only 79 and 62 of them, respectively. Our contact

  10. Accurate protein structure modeling using sparse NMR data and homologous structure information.

    PubMed

    Thompson, James M; Sgourakis, Nikolaos G; Liu, Gaohua; Rossi, Paolo; Tang, Yuefeng; Mills, Jeffrey L; Szyperski, Thomas; Montelione, Gaetano T; Baker, David

    2012-06-19

    While information from homologous structures plays a central role in X-ray structure determination by molecular replacement, such information is rarely used in NMR structure determination because it can be incorrect, both locally and globally, when evolutionary relationships are inferred incorrectly or there has been considerable evolutionary structural divergence. Here we describe a method that allows robust modeling of protein structures of up to 225 residues by combining (1)H(N), (13)C, and (15)N backbone and (13)Cβ chemical shift data, distance restraints derived from homologous structures, and a physically realistic all-atom energy function. Accurate models are distinguished from inaccurate models generated using incorrect sequence alignments by requiring that (i) the all-atom energies of models generated using the restraints are lower than models generated in unrestrained calculations and (ii) the low-energy structures converge to within 2.0 Å backbone rmsd over 75% of the protein. Benchmark calculations on known structures and blind targets show that the method can accurately model protein structures, even with very remote homology information, to a backbone rmsd of 1.2-1.9 Å relative to the conventional determined NMR ensembles and of 0.9-1.6 Å relative to X-ray structures for well-defined regions of the protein structures. This approach facilitates the accurate modeling of protein structures using backbone chemical shift data without need for side-chain resonance assignments and extensive analysis of NOESY cross-peak assignments.

  11. Accurate Filtering of Privacy-Sensitive Information in Raw Genomic Data.

    PubMed

    Decouchant, Jérémie; Fernandes, Maria; Völp, Marcus; Couto, Francisco M; Esteves-Veríssimo, Paulo

    2018-04-13

    Sequencing thousands of human genomes has enabled breakthroughs in many areas, among them precision medicine, the study of rare diseases, and forensics. However, mass collection of such sensitive data entails enormous risks if not protected to the highest standards. In this article, we follow the position and argue that post-alignment privacy is not enough and that data should be automatically protected as early as possible in the genomics workflow, ideally immediately after the data is produced. We show that a previous approach for filtering short reads cannot extend to long reads and present a novel filtering approach that classifies raw genomic data (i.e., whose location and content is not yet determined) into privacy-sensitive (i.e., more affected by a successful privacy attack) and non-privacy-sensitive information. Such a classification allows the fine-grained and automated adjustment of protective measures to mitigate the possible consequences of exposure, in particular when relying on public clouds. We present the first filter that can be indistinctly applied to reads of any length, i.e., making it usable with any recent or future sequencing technologies. The filter is accurate, in the sense that it detects all known sensitive nucleotides except those located in highly variable regions (less than 10 nucleotides remain undetected per genome instead of 100,000 in previous works). It has far less false positives than previously known methods (10% instead of 60%) and can detect sensitive nucleotides despite sequencing errors (86% detected instead of 56% with 2% of mutations). Finally, practical experiments demonstrate high performance, both in terms of throughput and memory consumption. Copyright © 2018. Published by Elsevier Inc.

  12. Toxicogenomics and Cancer Susceptibility: Advances with Next-Generation Sequencing

    PubMed Central

    Ning, Baitang; Su, Zhenqiang; Mei, Nan; Hong, Huixiao; Deng, Helen; Shi, Leming; Fuscoe, James C.; Tolleson, William H.

    2017-01-01

    The aim of this review is to comprehensively summarize the recent achievements in the field of toxicogenomics and cancer research regarding genetic-environmental interactions in carcinogenesis and detection of genetic aberrations in cancer genomes by next-generation sequencing technology. Cancer is primarily a genetic disease in which genetic factors and environmental stimuli interact to cause genetic and epigenetic aberrations in human cells. Mutations in the germline act as either high-penetrance alleles that strongly increase the risk of cancer development, or as low-penetrance alleles that mildly change an individual’s susceptibility to cancer. Somatic mutations, resulting from either DNA damage induced by exposure to environmental mutagens or from spontaneous errors in DNA replication or repair are involved in the development or progression of the cancer. Induced or spontaneous changes in the epigenome may also drive carcinogenesis. Advances in next-generation sequencing technology provide us opportunities to accurately, economically, and rapidly identify genetic variants, somatic mutations, gene expression profiles, and epigenetic alterations with single-base resolution. Whole genome sequencing, whole exome sequencing, and RNA sequencing of paired cancer and adjacent normal tissue present a comprehensive picture of the cancer genome. These new findings should benefit public health by providing insights in understanding cancer biology, and in improving cancer diagnosis and therapy. PMID:24875441

  13. Impacts of visuomotor sequence learning methods on speed and accuracy: Starting over from the beginning or from the point of error.

    PubMed

    Tanaka, Kanji; Watanabe, Katsumi

    2016-02-01

    The present study examined whether sequence learning led to more accurate and shorter performance time if people who are learning a sequence start over from the beginning when they make an error (i.e., practice the whole sequence) or only from the point of error (i.e., practice a part of the sequence). We used a visuomotor sequence learning paradigm with a trial-and-error procedure. In Experiment 1, we found fewer errors, and shorter performance time for those who restarted their performance from the beginning of the sequence as compared to those who restarted from the point at which an error occurred, indicating better learning of spatial and motor representations of the sequence. This might be because the learned elements were repeated when the next performance started over from the beginning. In subsequent experiments, we increased the occasions for the repetitions of learned elements by modulating the number of fresh start points in the sequence after errors. The results showed that fewer fresh start points were likely to lead to fewer errors and shorter performance time, indicating that the repetitions of learned elements enabled participants to develop stronger spatial and motor representations of the sequence. Thus, a single or two fresh start points in the sequence (i.e., starting over only from the beginning or from the beginning or midpoint of the sequence after errors) is likely to lead to more accurate and faster performance. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Nanopore sequencing technology and tools for genome assembly: computational analysis of the current state, bottlenecks and future directions.

    PubMed

    Senol Cali, Damla; Kim, Jeremie S; Ghose, Saugata; Alkan, Can; Mutlu, Onur

    2018-04-02

    Nanopore sequencing technology has the potential to render other sequencing technologies obsolete with its ability to generate long reads and provide portability. However, high error rates of the technology pose a challenge while generating accurate genome assemblies. The tools used for nanopore sequence analysis are of critical importance, as they should overcome the high error rates of the technology. Our goal in this work is to comprehensively analyze current publicly available tools for nanopore sequence analysis to understand their advantages, disadvantages and performance bottlenecks. It is important to understand where the current tools do not perform well to develop better tools. To this end, we (1) analyze the multiple steps and the associated tools in the genome assembly pipeline using nanopore sequence data, and (2) provide guidelines for determining the appropriate tools for each step. Based on our analyses, we make four key observations: (1) the choice of the tool for basecalling plays a critical role in overcoming the high error rates of nanopore sequencing technology. (2) Read-to-read overlap finding tools, GraphMap and Minimap, perform similarly in terms of accuracy. However, Minimap has a lower memory usage, and it is faster than GraphMap. (3) There is a trade-off between accuracy and performance when deciding on the appropriate tool for the assembly step. The fast but less accurate assembler Miniasm can be used for quick initial assembly, and further polishing can be applied on top of it to increase the accuracy, which leads to faster overall assembly. (4) The state-of-the-art polishing tool, Racon, generates high-quality consensus sequences while providing a significant speedup over another polishing tool, Nanopolish. We analyze various combinations of different tools and expose the trade-offs between accuracy, performance, memory usage and scalability. We conclude that our observations can guide researchers and practitioners in making conscious

  15. Characterizing the D2 statistic: word matches in biological sequences.

    PubMed

    Forêt, Sylvain; Wilson, Susan R; Burden, Conrad J

    2009-01-01

    Word matches are often used in sequence comparison methods, either as a measure of sequence similarity or in the first search steps of algorithms such as BLAST or BLAT. The D2 statistic is the number of matches of words of k letters between two sequences. Recent advances have been made in the characterization of this statistic and in the approximation of its distribution. Here, these results are extended to the case of approximate word matches. We compute the exact value of the variance of the D2 statistic for the case of a uniform letter distribution, and introduce a method to provide accurate approximations of the variance in the remaining cases. This enables the distribution of D2 to be approximated for typical situations arising in biological research. We apply these results to the identification of cis-regulatory modules, and show that this method detects such sequences with a high accuracy. The ability to approximate the distribution of D2 for both exact and approximate word matches will enable the use of this statistic in a more precise manner for sequence comparison, database searches, and identification of transcription factor binding sites.

  16. Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers.

    PubMed

    Williams-Woods, Jacquelina; González-Escalona, Narjol; Burkhardt, William

    2011-12-01

    Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89-150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ≥95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays. Published by Elsevier B.V.

  17. Microorganisms with Novel Dissimilatory (Bi)Sulfite Reductase Genes Are Widespread and Part of the Core Microbiota in Low-Sulfate Peatlands ▿ †

    PubMed Central

    Steger, Doris; Wentrup, Cecilia; Braunegger, Christina; Deevong, Pinsurang; Hofer, Manuel; Richter, Andreas; Baranyi, Christian; Pester, Michael; Wagner, Michael; Loy, Alexander

    2011-01-01

    Peatlands of the Lehstenbach catchment (Germany) house as-yet-unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic of microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a 6-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented “core” members (up to 1% to 1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparisons of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance of ∼1 to 400 km) identified that one Syntrophobacter-related and nine novel dsrAB lineages are widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-based DGGE data were not correlated with geographic distance but could be explained largely by soil pH and wetland type, implying that the distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by local environmental conditions. PMID:21169452

  18. A Novel Center Star Multiple Sequence Alignment Algorithm Based on Affine Gap Penalty and K-Band

    NASA Astrophysics Data System (ADS)

    Zou, Quan; Shan, Xiao; Jiang, Yi

    Multiple sequence alignment is one of the most important topics in computational biology, but it cannot deal with the large data so far. As the development of copy-number variant(CNV) and Single Nucleotide Polymorphisms(SNP) research, many researchers want to align numbers of similar sequences for detecting CNV and SNP. In this paper, we propose a novel multiple sequence alignment algorithm based on affine gap penalty and k-band. It can align more quickly and accurately, that will be helpful for mining CNV and SNP. Experiments prove the performance of our algorithm.

  19. A statistical method for assessing peptide identification confidence in accurate mass and time tag proteomics

    PubMed Central

    Stanley, Jeffrey R.; Adkins, Joshua N.; Slysz, Gordon W.; Monroe, Matthew E.; Purvine, Samuel O.; Karpievitch, Yuliya V.; Anderson, Gordon A.; Smith, Richard D.; Dabney, Alan R.

    2011-01-01

    Current algorithms for quantifying peptide identification confidence in the accurate mass and time (AMT) tag approach assume that the AMT tags themselves have been correctly identified. However, there is uncertainty in the identification of AMT tags, as this is based on matching LC-MS/MS fragmentation spectra to peptide sequences. In this paper, we incorporate confidence measures for the AMT tag identifications into the calculation of probabilities for correct matches to an AMT tag database, resulting in a more accurate overall measure of identification confidence for the AMT tag approach. The method is referred to as Statistical Tools for AMT tag Confidence (STAC). STAC additionally provides a Uniqueness Probability (UP) to help distinguish between multiple matches to an AMT tag and a method to calculate an overall false discovery rate (FDR). STAC is freely available for download as both a command line and a Windows graphical application. PMID:21692516

  20. [Identification of common medicinal snakes in medicated liquor of Guangdong by COI barcode sequence].

    PubMed

    Liao, Jing; Chao, Zhi; Zhang, Liang

    2013-11-01

    To identify the common snakes in medicated liquor of Guangdong using COI barcode sequence,and to test the feasibility. The COI barcode sequences of collected medicinal snakes were amplified and sequenced. The sequences combined with the data from GenBank were analyzed for divergence and building a neighbor-joining(NJ) tree with MEGA 5.0. The genetic distance and NJ tree demonstrated that there were 241 variable sites in these species, and the average (A + T) content of 56.2% was higher than the average (G + C) content of 43.7%. The maximum interspecific genetic distance was 0.2568, and the minimum was 0. 1519. In the NJ tree,each species formed a monophyletic clade with bootstrap supports of 100%. DNA barcoding identification method based on the COI sequence is accurate and can be applied to identify the common medicinal snakes.

  1. The sequence of sequencers: The history of sequencing DNA.

    PubMed

    Heather, James M; Chain, Benjamin

    2016-01-01

    Determining the order of nucleic acid residues in biological samples is an integral component of a wide variety of research applications. Over the last fifty years large numbers of researchers have applied themselves to the production of techniques and technologies to facilitate this feat, sequencing DNA and RNA molecules. This time-scale has witnessed tremendous changes, moving from sequencing short oligonucleotides to millions of bases, from struggling towards the deduction of the coding sequence of a single gene to rapid and widely available whole genome sequencing. This article traverses those years, iterating through the different generations of sequencing technology, highlighting some of the key discoveries, researchers, and sequences along the way. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Implicit learning of non-spatial sequences in schizophrenia

    PubMed Central

    MARVEL, CHERIE L.; SCHWARTZ, BARBARA L.; HOWARD, DARLENE V.; HOWARD, JAMES H.

    2006-01-01

    Recent studies have reported abnormal implicit learning of sequential patterns in patients with schizophrenia. Because these studies were based on visuospatial cues, the question remained whether patients were impaired simply due to the demands of spatial processing. This study examined implicit sequence learning in 24 patients with schizophrenia and 24 healthy controls using a non-spatial variation of the serial reaction time test (SRT) in which pattern stimuli alternated with random stimuli on every other trial. Both groups showed learning by responding faster and more accurately to pattern trials than to random trials. Patients, however, showed a smaller magnitude of sequence learning. Both groups were unable to demonstrate explicit knowledge of the nature of the pattern, confirming that learning occurred without awareness. Clinical variables were not correlated with the patients' learning deficits. Patients with schizophrenia have a decreased ability to develop sensitivity to regularly occurring sequences of events within their environment. This type of deficit may affect an array of cognitive and motor functions that rely on the perception of event regularity. PMID:16248901

  3. Sequencing ebola and marburg viruses genomes using microarrays.

    PubMed

    Hardick, Justin; Woelfel, Roman; Gardner, Warren; Ibrahim, Sofi

    2016-08-01

    Periodic outbreaks of Ebola and Marburg hemorrhagic fevers have occurred in Africa over the past four decades with case fatality rates reaching as high as 90%. The latest Ebola outbreak in West Africa in 2014 raised concerns that these infections can spread across continents and pose serious health risks. Early and accurate identification of the causative agents is necessary to contain outbreaks. In this report, we describe sequencing-by-hybridization (SBH) technique using high density microarrays to identify Ebola and Marburg viruses. The microarrays were designed to interrogate the sequences of entire viral genomes, and were evaluated with three species of Ebolavirus (Reston, Sudan, and Zaire), and three strains of Marburgvirus (Angola, Musoke, and Ravn). The results showed that the consensus sequences generated with four or more hybridizations had 92.1-98.9% accuracy over 95-99% of the genomes. Additionally, with SBH microarrays it was possible to distinguish between different strains of the Lake Victoria Marburgvirus. J. Med. Virol. 88:1303-1308, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Applications of nuclear magnetic resonance in paediatric surgery: magnetic resonance cholangiopancreatography and new sequences.

    PubMed

    Ragazzi, S; Vanzulli, A; Del Maschio, A; Tomaselli, V; Dell' Agnola, C A

    2007-12-01

    The aim of the study was to evaluate fast magnetic resonance cholangio-pancreatography (MRCP) sequences as an alternative and safe investigation method for neonatal and children's pancreaticobiliary diseases. Between January 2000 and December 2000, five children (age: 1 month 14 years; mean: 7 years) affected by pancreaticobiliary diseases or already operated for biliary pathologies were studied. Patients were evaluated by 1.5 T magnet single shot T2-weighted sequences (1 image per s, TR = infinite, TE = 150-180 ms). T1-weighted conventional sequences were obtained to study parenchymal tissue. No patient needed general anaesthetic. Only in one case was sedation necessary. Fast MRCP sequences provided very precise information on biliary tract anatomy. They revealed the intra and extrahepatic bile ducts, the gallbladder, the common bile duct and the bilio-pancreatic junction in all cases investigated. MRCP allowed us to evaluate Roux-en-Y type bilio-enteric anastomosis as accurately as percutaneous transhepatic cholangiography (PTC). In addition MRCP was the only reliable study in evaluating Roux-en-Y type anastomosis where ultrasonography (US) and endoscopic retrograde cholangiography (ERCP) could not be used. In conclusion MRCP is an accurate and non-invasive method with which to investigate the anatomy of the pancreaticobiliary tract in children. It could become the investigation of choice after US in the case of biliary and pancreatic diseases.

  5. Genome Sequencing of 18 Francisella Strains To Aid in Assay Development and Testing

    DOE PAGES

    Johnson, Shannon L.; Daligault, Hajnalka E.; Davenport, Karen W.; ...

    2015-04-30

    Francisella tularensis is a highly infectious bacterium that has the potential of causing high fatality rates if infections are untreated. To aid in the development of rapid and accurate detection assays, we have sequenced and annotated the genomes of 18 F. tularensis and Francisella philomiragia strains.

  6. Protein sequencing via nanopore based devices: a nanofluidics perspective

    NASA Astrophysics Data System (ADS)

    Chinappi, Mauro; Cecconi, Fabio

    2018-05-01

    Proteins perform a huge number of central functions in living organisms, thus all the new techniques allowing their precise, fast and accurate characterization at single-molecule level certainly represent a burst in proteomics with important biomedical impact. In this review, we describe the recent progresses in the developing of nanopore based devices for protein sequencing. We start with a critical analysis of the main technical requirements for nanopore protein sequencing, summarizing some ideas and methodologies that have recently appeared in the literature. In the last sections, we focus on the physical modelling of the transport phenomena occurring in nanopore based devices. The multiscale nature of the problem is discussed and, in this respect, some of the main possible computational approaches are illustrated.

  7. Software for rapid time dependent ChIP-sequencing analysis (TDCA).

    PubMed

    Myschyshyn, Mike; Farren-Dai, Marco; Chuang, Tien-Jui; Vocadlo, David

    2017-11-25

    Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and associated methods are widely used to define the genome wide distribution of chromatin associated proteins, post-translational epigenetic marks, and modifications found on DNA bases. An area of emerging interest is to study time dependent changes in the distribution of such proteins and marks by using serial ChIP-seq experiments performed in a time resolved manner. Despite such time resolved studies becoming increasingly common, software to facilitate analysis of such data in a robust automated manner is limited. We have designed software called Time-Dependent ChIP-Sequencing Analyser (TDCA), which is the first program to automate analysis of time-dependent ChIP-seq data by fitting to sigmoidal curves. We provide users with guidance for experimental design of TDCA for modeling of time course (TC) ChIP-seq data using two simulated data sets. Furthermore, we demonstrate that this fitting strategy is widely applicable by showing that automated analysis of three previously published TC data sets accurately recapitulates key findings reported in these studies. Using each of these data sets, we highlight how biologically relevant findings can be readily obtained by exploiting TDCA to yield intuitive parameters that describe behavior at either a single locus or sets of loci. TDCA enables customizable analysis of user input aligned DNA sequencing data, coupled with graphical outputs in the form of publication-ready figures that describe behavior at either individual loci or sets of loci sharing common traits defined by the user. TDCA accepts sequencing data as standard binary alignment map (BAM) files and loci of interest in browser extensible data (BED) file format. TDCA accurately models the number of sequencing reads, or coverage, at loci from TC ChIP-seq studies or conceptually related TC sequencing experiments. TC experiments are reduced to intuitive parametric values that facilitate biologically

  8. Accurate prediction of secondary metabolite gene clusters in filamentous fungi.

    PubMed

    Andersen, Mikael R; Nielsen, Jakob B; Klitgaard, Andreas; Petersen, Lene M; Zachariasen, Mia; Hansen, Tilde J; Blicher, Lene H; Gotfredsen, Charlotte H; Larsen, Thomas O; Nielsen, Kristian F; Mortensen, Uffe H

    2013-01-02

    Biosynthetic pathways of secondary metabolites from fungi are currently subject to an intense effort to elucidate the genetic basis for these compounds due to their large potential within pharmaceutics and synthetic biochemistry. The preferred method is methodical gene deletions to identify supporting enzymes for key synthases one cluster at a time. In this study, we design and apply a DNA expression array for Aspergillus nidulans in combination with legacy data to form a comprehensive gene expression compendium. We apply a guilt-by-association-based analysis to predict the extent of the biosynthetic clusters for the 58 synthases active in our set of experimental conditions. A comparison with legacy data shows the method to be accurate in 13 of 16 known clusters and nearly accurate for the remaining 3 clusters. Furthermore, we apply a data clustering approach, which identifies cross-chemistry between physically separate gene clusters (superclusters), and validate this both with legacy data and experimentally by prediction and verification of a supercluster consisting of the synthase AN1242 and the prenyltransferase AN11080, as well as identification of the product compound nidulanin A. We have used A. nidulans for our method development and validation due to the wealth of available biochemical data, but the method can be applied to any fungus with a sequenced and assembled genome, thus supporting further secondary metabolite pathway elucidation in the fungal kingdom.

  9. NNLOPS accurate associated HW production

    NASA Astrophysics Data System (ADS)

    Astill, William; Bizon, Wojciech; Re, Emanuele; Zanderighi, Giulia

    2016-06-01

    We present a next-to-next-to-leading order accurate description of associated HW production consistently matched to a parton shower. The method is based on reweighting events obtained with the HW plus one jet NLO accurate calculation implemented in POWHEG, extended with the MiNLO procedure, to reproduce NNLO accurate Born distributions. Since the Born kinematics is more complex than the cases treated before, we use a parametrization of the Collins-Soper angles to reduce the number of variables required for the reweighting. We present phenomenological results at 13 TeV, with cuts suggested by the Higgs Cross section Working Group.

  10. A robust recognition and accurate locating method for circular coded diagonal target

    NASA Astrophysics Data System (ADS)

    Bao, Yunna; Shang, Yang; Sun, Xiaoliang; Zhou, Jiexin

    2017-10-01

    As a category of special control points which can be automatically identified, artificial coded targets have been widely developed in the field of computer vision, photogrammetry, augmented reality, etc. In this paper, a new circular coded target designed by RockeTech technology Corp. Ltd is analyzed and studied, which is called circular coded diagonal target (CCDT). A novel detection and recognition method with good robustness is proposed in the paper, and implemented on Visual Studio. In this algorithm, firstly, the ellipse features of the center circle are used for rough positioning. Then, according to the characteristics of the center diagonal target, a circular frequency filter is designed to choose the correct center circle and eliminates non-target noise. The precise positioning of the coded target is done by the correlation coefficient fitting extreme value method. Finally, the coded target recognition is achieved by decoding the binary sequence in the outer ring of the extracted target. To test the proposed algorithm, this paper has carried out simulation experiments and real experiments. The results show that the CCDT recognition and accurate locating method proposed in this paper can robustly recognize and accurately locate the targets in complex and noisy background.

  11. Muver, a computational framework for accurately calling accumulated mutations.

    PubMed

    Burkholder, Adam B; Lujan, Scott A; Lavender, Christopher A; Grimm, Sara A; Kunkel, Thomas A; Fargo, David C

    2018-05-09

    Identification of mutations from next-generation sequencing data typically requires a balance between sensitivity and accuracy. This is particularly true of DNA insertions and deletions (indels), that can impart significant phenotypic consequences on cells but are harder to call than substitution mutations from whole genome mutation accumulation experiments. To overcome these difficulties, we present muver, a computational framework that integrates established bioinformatics tools with novel analytical methods to generate mutation calls with the extremely low false positive rates and high sensitivity required for accurate mutation rate determination and comparison. Muver uses statistical comparison of ancestral and descendant allelic frequencies to identify variant loci and assigns genotypes with models that include per-sample assessments of sequencing errors by mutation type and repeat context. Muver identifies maximally parsimonious mutation pathways that connect these genotypes, differentiating potential allelic conversion events and delineating ambiguities in mutation location, type, and size. Benchmarking with a human gold standard father-son pair demonstrates muver's sensitivity and low false positive rates. In DNA mismatch repair (MMR) deficient Saccharomyces cerevisiae, muver detects multi-base deletions in homopolymers longer than the replicative polymerase footprint at rates greater than predicted for sequential single-base deletions, implying a novel multi-repeat-unit slippage mechanism. Benchmarking results demonstrate the high accuracy and sensitivity achieved with muver, particularly for indels, relative to available tools. Applied to an MMR-deficient Saccharomyces cerevisiae system, muver mutation calls facilitate mechanistic insights into DNA replication fidelity.

  12. Maturity onset diabetes of youth (MODY) in Turkish children: sequence analysis of 11 causative genes by next generation sequencing.

    PubMed

    Ağladıoğlu, Sebahat Yılmaz; Aycan, Zehra; Çetinkaya, Semra; Baş, Veysel Nijat; Önder, Aşan; Peltek Kendirci, Havva Nur; Doğan, Haldun; Ceylaner, Serdar

    2016-04-01

    Maturity-onset diabetes of the youth (MODY), is a genetically and clinically heterogeneous group of diseasesand is often misdiagnosed as type 1 or type 2 diabetes. The aim of this study is to investigate both novel and proven mutations of 11 MODY genes in Turkish children by using targeted next generation sequencing. A panel of 11 MODY genes were screened in 43 children with MODY diagnosed by clinical criterias. Studies of index cases was done with MISEQ-ILLUMINA, and family screenings and confirmation studies of mutations was done by Sanger sequencing. We identified 28 (65%) point mutations among 43 patients. Eighteen patients have GCK mutations, four have HNF1A, one has HNF4A, one has HNF1B, two have NEUROD1, one has PDX1 gene variations and one patient has both HNF1A and HNF4A heterozygote mutations. This is the first study including molecular studies of 11 MODY genes in Turkish children. GCK is the most frequent type of MODY in our study population. Very high frequency of novel mutations (42%) in our study population, supports that in heterogenous disorders like MODY sequence analysis provides rapid, cost effective and accurate genetic diagnosis.

  13. Sequencing small genomic targets with high efficiency and extreme accuracy

    PubMed Central

    Schmitt, Michael W.; Fox, Edward J.; Prindle, Marc J.; Reid-Bayliss, Kate S.; True, Lawrence D.; Radich, Jerald P.; Loeb, Lawrence A.

    2015-01-01

    The detection of minority variants in mixed samples demands methods for enrichment and accurate sequencing of small genomic intervals. We describe an efficient approach based on sequential rounds of hybridization with biotinylated oligonucleotides, enabling more than one-million fold enrichment of genomic regions of interest. In conjunction with error correcting double-stranded molecular tags, our approach enables the quantification of mutations in individual DNA molecules. PMID:25849638

  14. An Internet-Accessible DNA Sequence Database for Identifying Fusaria from Human and Animal Infections

    USDA-ARS?s Scientific Manuscript database

    Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated wi...

  15. Accurate quantum Z rotations with less magic

    NASA Astrophysics Data System (ADS)

    Landahl, Andrew; Cesare, Chris

    2013-03-01

    We present quantum protocols for executing arbitrarily accurate π /2k rotations of a qubit about its Z axis. Unlike reduced instruction set computing (RISC) protocols which use a two-step process of synthesizing high-fidelity ``magic'' states from which T = Z (π / 4) gates can be teleported and then compiling a sequence of adaptive stabilizer operations and T gates to approximate Z (π /2k) , our complex instruction set computing (CISC) protocol distills magic states for the Z (π /2k) gates directly. Replacing this two-step process with a single step results in substantial reductions in the number of gates needed. The key to our construction is a family of shortened quantum Reed-Muller codes of length 2 k + 2 - 1 , whose distillation threshold shrinks with k but is greater than 0.85% for k <= 6 . AJL and CC were supported in part by the Laboratory Directed Research and Development program at Sandia National Laboratories. Sandia National Laboratories is a multi-program laboratory managed and operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin Corporation, for the U.S. Department of Energy's National Nuclear Security Administration under contract DE-AC04-94AL85000.

  16. Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1.

    PubMed

    Charpentier, Elena; Garnaud, Cécile; Wintenberger, Claire; Bailly, Sébastien; Murat, Jean-Benjamin; Rendu, John; Pavese, Patricia; Drouet, Thibault; Augier, Caroline; Malvezzi, Paolo; Thiébaut-Bertrand, Anne; Mallaret, Marie-Reine; Epaulard, Olivier; Cornet, Muriel; Larrat, Sylvie; Maubon, Danièle

    2017-08-01

    Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus.

  17. Added Value of Next-Generation Sequencing for Multilocus Sequence Typing Analysis of a Pneumocystis jirovecii Pneumonia Outbreak1

    PubMed Central

    Charpentier, Elena; Garnaud, Cécile; Wintenberger, Claire; Bailly, Sébastien; Murat, Jean-Benjamin; Rendu, John; Pavese, Patricia; Drouet, Thibault; Augier, Caroline; Malvezzi, Paolo; Thiébaut-Bertrand, Anne; Mallaret, Marie-Reine; Epaulard, Olivier; Cornet, Muriel; Larrat, Sylvie

    2017-01-01

    Pneumocystis jirovecii is a major threat for immunocompromised patients, and clusters of pneumocystis pneumonia (PCP) have been increasingly described in transplant units during the past decade. Exploring an outbreak transmission network requires complementary spatiotemporal and strain-typing approaches. We analyzed a PCP outbreak and demonstrated the added value of next-generation sequencing (NGS) for the multilocus sequence typing (MLST) study of P. jirovecii strains. Thirty-two PCP patients were included. Among the 12 solid organ transplant patients, 5 shared a major and unique genotype that was also found as a minor strain in a sixth patient. A transmission map analysis strengthened the suspicion of nosocomial acquisition of this strain for the 6 patients. NGS-MLST enables accurate determination of subpopulation, which allowed excluding other patients from the transmission network. NGS-MLST genotyping approach was essential to deciphering this outbreak. This innovative approach brings new insights for future epidemiologic studies on this uncultivable opportunistic fungus. PMID:28726611

  18. Kickoff to Conflict: A Sequence Analysis of Intra-State Conflict-Preceding Event Structures

    PubMed Central

    D'Orazio, Vito; Yonamine, James E.

    2015-01-01

    While many studies have suggested or assumed that the periods preceding the onset of intra-state conflict are similar across time and space, few have empirically tested this proposition. Using the Integrated Crisis Early Warning System's domestic event data in Asia from 1998–2010, we subject this proposition to empirical analysis. We code the similarity of government-rebel interactions in sequences preceding the onset of intra-state conflict to those preceding further periods of peace using three different metrics: Euclidean, Levenshtein, and mutual information. These scores are then used as predictors in a bivariate logistic regression to forecast whether we are likely to observe conflict in neither, one, or both of the states. We find that our model accurately classifies cases where both sequences precede peace, but struggles to distinguish between cases in which one sequence escalates to conflict and where both sequences escalate to conflict. These findings empirically suggest that generalizable patterns exist between event sequences that precede peace. PMID:25951105

  19. Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study.

    PubMed

    Jiang, Haojun; Xie, Yifan; Li, Xuchao; Ge, Huijuan; Deng, Yongqiang; Mu, Haofang; Feng, Xiaoli; Yin, Lu; Du, Zhou; Chen, Fang; He, Nongyue

    2016-01-01

    Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future.

  20. Efficient and accurate causal inference with hidden confounders from genome-transcriptome variation data

    PubMed Central

    2017-01-01

    Mapping gene expression as a quantitative trait using whole genome-sequencing and transcriptome analysis allows to discover the functional consequences of genetic variation. We developed a novel method and ultra-fast software Findr for higly accurate causal inference between gene expression traits using cis-regulatory DNA variations as causal anchors, which improves current methods by taking into consideration hidden confounders and weak regulations. Findr outperformed existing methods on the DREAM5 Systems Genetics challenge and on the prediction of microRNA and transcription factor targets in human lymphoblastoid cells, while being nearly a million times faster. Findr is publicly available at https://github.com/lingfeiwang/findr. PMID:28821014

  1. Swarm intelligence in bioinformatics: methods and implementations for discovering patterns of multiple sequences.

    PubMed

    Cui, Zhihua; Zhang, Yi

    2014-02-01

    As a promising and innovative research field, bioinformatics has attracted increasing attention recently. Beneath the enormous number of open problems in this field, one fundamental issue is about the accurate and efficient computational methodology that can deal with tremendous amounts of data. In this paper, we survey some applications of swarm intelligence to discover patterns of multiple sequences. To provide a deep insight, ant colony optimization, particle swarm optimization, artificial bee colony and artificial fish swarm algorithm are selected, and their applications to multiple sequence alignment and motif detecting problem are discussed.

  2. High-Accuracy HLA Type Inference from Whole-Genome Sequencing Data Using Population Reference Graphs.

    PubMed

    Dilthey, Alexander T; Gourraud, Pierre-Antoine; Mentzer, Alexander J; Cereb, Nezih; Iqbal, Zamin; McVean, Gil

    2016-10-01

    Genetic variation at the Human Leucocyte Antigen (HLA) genes is associated with many autoimmune and infectious disease phenotypes, is an important element of the immunological distinction between self and non-self, and shapes immune epitope repertoires. Determining the allelic state of the HLA genes (HLA typing) as a by-product of standard whole-genome sequencing data would therefore be highly desirable and enable the immunogenetic characterization of samples in currently ongoing population sequencing projects. Extensive hyperpolymorphism and sequence similarity between the HLA genes, however, pose problems for accurate read mapping and make HLA type inference from whole-genome sequencing data a challenging problem. We describe how to address these challenges in a Population Reference Graph (PRG) framework. First, we construct a PRG for 46 (mostly HLA) genes and pseudogenes, their genomic context and their characterized sequence variants, integrating a database of over 10,000 known allele sequences. Second, we present a sequence-to-PRG paired-end read mapping algorithm that enables accurate read mapping for the HLA genes. Third, we infer the most likely pair of underlying alleles at G group resolution from the IMGT/HLA database at each locus, employing a simple likelihood framework. We show that HLA*PRG, our algorithm, outperforms existing methods by a wide margin. We evaluate HLA*PRG on six classical class I and class II HLA genes (HLA-A, -B, -C, -DQA1, -DQB1, -DRB1) and on a set of 14 samples (3 samples with 2 x 100bp, 11 samples with 2 x 250bp Illumina HiSeq data). Of 158 alleles tested, we correctly infer 157 alleles (99.4%). We also identify and re-type two erroneous alleles in the original validation data. We conclude that HLA*PRG for the first time achieves accuracies comparable to gold-standard reference methods from standard whole-genome sequencing data, though high computational demands (currently ~30-250 CPU hours per sample) remain a significant

  3. High-Accuracy HLA Type Inference from Whole-Genome Sequencing Data Using Population Reference Graphs

    PubMed Central

    Dilthey, Alexander T.; Gourraud, Pierre-Antoine; McVean, Gil

    2016-01-01

    Genetic variation at the Human Leucocyte Antigen (HLA) genes is associated with many autoimmune and infectious disease phenotypes, is an important element of the immunological distinction between self and non-self, and shapes immune epitope repertoires. Determining the allelic state of the HLA genes (HLA typing) as a by-product of standard whole-genome sequencing data would therefore be highly desirable and enable the immunogenetic characterization of samples in currently ongoing population sequencing projects. Extensive hyperpolymorphism and sequence similarity between the HLA genes, however, pose problems for accurate read mapping and make HLA type inference from whole-genome sequencing data a challenging problem. We describe how to address these challenges in a Population Reference Graph (PRG) framework. First, we construct a PRG for 46 (mostly HLA) genes and pseudogenes, their genomic context and their characterized sequence variants, integrating a database of over 10,000 known allele sequences. Second, we present a sequence-to-PRG paired-end read mapping algorithm that enables accurate read mapping for the HLA genes. Third, we infer the most likely pair of underlying alleles at G group resolution from the IMGT/HLA database at each locus, employing a simple likelihood framework. We show that HLA*PRG, our algorithm, outperforms existing methods by a wide margin. We evaluate HLA*PRG on six classical class I and class II HLA genes (HLA-A, -B, -C, -DQA1, -DQB1, -DRB1) and on a set of 14 samples (3 samples with 2 x 100bp, 11 samples with 2 x 250bp Illumina HiSeq data). Of 158 alleles tested, we correctly infer 157 alleles (99.4%). We also identify and re-type two erroneous alleles in the original validation data. We conclude that HLA*PRG for the first time achieves accuracies comparable to gold-standard reference methods from standard whole-genome sequencing data, though high computational demands (currently ~30–250 CPU hours per sample) remain a significant

  4. Enabling multiplexed testing of pooled donor cells through whole-genome sequencing.

    PubMed

    Chan, Yingleong; Chan, Ying Kai; Goodman, Daniel B; Guo, Xiaoge; Chavez, Alejandro; Lim, Elaine T; Church, George M

    2018-04-19

    We describe a method that enables the multiplex screening of a pool of many different donor cell lines. Our method accurately predicts each donor proportion from the pool without requiring the use of unique DNA barcodes as markers of donor identity. Instead, we take advantage of common single nucleotide polymorphisms, whole-genome sequencing, and an algorithm to calculate the proportions from the sequencing data. By testing using simulated and real data, we showed that our method robustly predicts the individual proportions from a mixed-pool of numerous donors, thus enabling the multiplexed testing of diverse donor cells en masse.More information is available at https://pgpresearch.med.harvard.edu/poolseq/.

  5. Next-Generation Sequencing: a Diagnostic One-Stop Shop for Hepatitis C?

    PubMed

    Poljak, Mario

    2016-10-01

    Before starting chronic hepatitis C treatment, the viral genotype/subtype has to be accurately determined and potentially coupled with drug resistance testing. Due to the high genetic variability of the hepatitis C virus, this can be a demanding task that can potentially be streamlined by viral whole-genome sequencing using next-generation sequencing as demonstrated by an article in this issue of the Journal of Clinical Microbiology by E. Thomson, C. L. C. Ip, A. Badhan, M. T. Christiansen, W. Adamson, et al. (J Clin Microbiol. 54:2455-2469, 2016, http://dx.doi.org/10.1128/JCM.00330-16). Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. Accurate Arabic Script Language/Dialect Classification

    DTIC Science & Technology

    2014-01-01

    Army Research Laboratory Accurate Arabic Script Language/Dialect Classification by Stephen C. Tratz ARL-TR-6761 January 2014 Approved for public...1197 ARL-TR-6761 January 2014 Accurate Arabic Script Language/Dialect Classification Stephen C. Tratz Computational and Information Sciences...Include area code) Standard Form 298 (Rev. 8/98) Prescribed by ANSI Std. Z39.18 January 2014 Final Accurate Arabic Script Language/Dialect Classification

  7. Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

    PubMed

    Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K P; Woo, Patrick C Y

    2015-10-22

    Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.

  8. Using pseudoalignment and base quality to accurately quantify microbial community composition

    PubMed Central

    Novembre, John

    2018-01-01

    Pooled DNA from multiple unknown organisms arises in a variety of contexts, for example microbial samples from ecological or human health research. Determining the composition of pooled samples can be difficult, especially at the scale of modern sequencing data and reference databases. Here we propose a novel method for taxonomic profiling in pooled DNA that combines the speed and low-memory requirements of k-mer based pseudoalignment with a likelihood framework that uses base quality information to better resolve multiply mapped reads. We apply the method to the problem of classifying 16S rRNA reads using a reference database of known organisms, a common challenge in microbiome research. Using simulations, we show the method is accurate across a variety of read lengths, with different length reference sequences, at different sample depths, and when samples contain reads originating from organisms absent from the reference. We also assess performance in real 16S data, where we reanalyze previous genetic association data to show our method discovers a larger number of quantitative trait associations than other widely used methods. We implement our method in the software Karp, for k-mer based analysis of read pools, to provide a novel combination of speed and accuracy that is uniquely suited for enhancing discoveries in microbial studies. PMID:29659582

  9. High-throughput sequencing reveals unprecedented diversities of Aspergillus species in outdoor air.

    PubMed

    Lee, S; An, C; Xu, S; Lee, S; Yamamoto, N

    2016-09-01

    This study used the Illumina MiSeq to analyse compositions and diversities of Aspergillus species in outdoor air. The seasonal air samplings were performed at two locations in Seoul, South Korea. The results showed the relative abundances of all Aspergillus species combined ranging from 0·20 to 18% and from 0·19 to 21% based on the number of the internal transcribed spacer 1 (ITS1) and β-tubulin (BenA) gene sequences respectively. Aspergillus fumigatus was the most dominant species with the mean relative abundances of 1·2 and 5·5% based on the number of the ITS1 and BenA sequences respectively. A total of 29 Aspergillus species were detected and identified down to the species rank, among which nine species were known opportunistic pathogens. Remarkably, eight of the nine pathogenic species were detected by either one of the two markers, suggesting the need of using multiple markers and/or primer pairs when the assessments are made based on the high-throughput sequencing. Due to diversity of species within the genus Aspergillus, the high-throughput sequencing was useful to characterize their compositions and diversities in outdoor air, which are thought to be difficult to be accurately characterized by conventional culture and/or Sanger sequencing-based techniques. Aspergillus is a diverse genus of fungi with more than 300 species reported in literature. Aspergillus is important since some species are known allergens and opportunistic human pathogens. Traditionally, growth-dependent methods have been used to detect Aspergillus species in air. However, these methods are limited in the number of isolates that can be analysed for their identities, resulting in inaccurate characterizations of Aspergillus diversities. This study used the high-throughput sequencing to explore Aspergillus diversities in outdoor, which are thought to be difficult to be accurately characterized by traditional growth-dependent techniques. © 2016 The Society for Applied Microbiology.

  10. Whole genome sequencing in the prevention and control of Staphylococcus aureus infection.

    PubMed

    Price, J R; Didelot, X; Crook, D W; Llewelyn, M J; Paul, J

    2013-01-01

    Staphylococcus aureus remains a leading cause of hospital-acquired infection but weaknesses inherent in currently available typing methods impede effective infection prevention and control. The high resolution offered by whole genome sequencing has the potential to revolutionise our understanding and management of S. aureus infection. To outline the practicalities of whole genome sequencing and discuss how it might shape future infection control practice. We review conventional typing methods and compare these with the potential offered by whole genome sequencing. In contrast with conventional methods, whole genome sequencing discriminates down to single nucleotide differences and allows accurate characterisation of transmission events and outbreaks and additionally provides information about the genetic basis of phenotypic characteristics, including antibiotic susceptibility and virulence. However, translating its potential into routine practice will depend on affordability, acceptable turnaround times and on creating a reliable standardised bioinformatic infrastructure. Whole genome sequencing has the potential to provide a universal test that facilitates outbreak investigation, enables the detection of emerging strains and predicts their clinical importance. Copyright © 2012 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  11. 3D knee segmentation based on three MRI sequences from different planes.

    PubMed

    Zhou, L; Chav, R; Cresson, T; Chartrand, G; de Guise, J

    2016-08-01

    In clinical practice, knee MRI sequences with 3.5~5 mm slice distance in sagittal, coronal, and axial planes are often requested for the knee examination since its acquisition is faster than high-resolution MRI sequence in a single plane, thereby reducing the probability of motion artifact. In order to take advantage of the three sequences from different planes, a 3D segmentation method based on the combination of three knee models obtained from the three sequences is proposed in this paper. In the method, the sub-segmentation is respectively performed with sagittal, coronal, and axial MRI sequence in the image coordinate system. With each sequence, an initial knee model is hierarchically deformed, and then the three deformed models are mapped to reference coordinate system defined by the DICOM standard and combined to obtain a patient-specific model. The experimental results verified that the three sub-segmentation results can complement each other, and their integration can compensate for the insufficiency of boundary information caused by 3.5~5 mm gap between consecutive slices. Therefore, the obtained patient-specific model is substantially more accurate than each sub-segmentation results.

  12. The sequence measurement system of the IR camera

    NASA Astrophysics Data System (ADS)

    Geng, Ai-hui; Han, Hong-xia; Zhang, Hai-bo

    2011-08-01

    program written by the verilog language combining the SignalTap tool on line observation can count the line numbers in one frame, pixel numbers in one line and meanwhile account the line offset and row offset of the image. Aiming at the complex sequence of the IR camera's output signal, the sequence measurement system of the IR camera accurately measures the sequence of the project applied camera, supplies the detailed sequence document to the continuous system such as image processing system and image transmission system and gives out the concrete parameters of the fval, lval, pixclk, line offset and row offset. The experiment shows that the sequence measurement system of the IR camera can get the precise sequence measurement result and works stably, laying foundation for the continuous system.

  13. Adenosine stress cardiovascular magnetic resonance with variable-density spiral pulse sequences accurately detects coronary artery disease: initial clinical evaluation.

    PubMed

    Salerno, Michael; Taylor, Angela; Yang, Yang; Kuruvilla, Sujith; Ragosta, Michael; Meyer, Craig H; Kramer, Christopher M

    2014-07-01

    Adenosine stress cardiovascular magnetic resonance perfusion imaging can be limited by motion-induced dark-rim artifacts, which may be mistaken for true perfusion abnormalities. A high-resolution variable-density spiral pulse sequence with a novel density compensation strategy has been shown to reduce dark-rim artifacts in first-pass perfusion imaging. We aimed to assess the clinical performance of adenosine stress cardiovascular magnetic resonance using this new perfusion sequence to detect obstructive coronary artery disease. Cardiovascular magnetic resonance perfusion imaging was performed during adenosine stress (140 μg/kg per minute) and at rest on a Siemens 1.5-T Avanto scanner in 41 subjects with chest pain scheduled for coronary angiography. Perfusion images were acquired during injection of 0.1 mmol/kg Gadolinium-diethylenetriaminepentacetate at 3 short-axis locations using a saturation recovery interleaved variable-density spiral pulse sequence. Significant stenosis was defined as >50% by quantitative coronary angiography. Two blinded reviewers evaluated the perfusion images for the presence of adenosine-induced perfusion abnormalities and assessed image quality using a 5-point scale (1 [poor] to 5 [excellent]). The prevalence of obstructive coronary artery disease by quantitative coronary angiography was 68%. The average sensitivity, specificity, and accuracy were 89%, 85%, and 88%, respectively, with a positive predictive value and negative predictive value of 93% and 79%, respectively. The average image quality score was 4.4±0.7, with only 1 study with more than mild dark-rim artifacts. There was good inter-reader reliability with a κ statistic of 0.67. Spiral adenosine stress cardiovascular magnetic resonance results in high diagnostic accuracy for the detection of obstructive coronary artery disease with excellent image quality and minimal dark-rim artifacts. © 2014 American Heart Association, Inc.

  14. Application of Stochastic Labeling with Random-Sequence Barcodes for Simultaneous Quantification and Sequencing of Environmental 16S rRNA Genes.

    PubMed

    Hoshino, Tatsuhiko; Inagaki, Fumio

    2017-01-01

    Next-generation sequencing (NGS) is a powerful tool for analyzing environmental DNA and provides the comprehensive molecular view of microbial communities. For obtaining the copy number of particular sequences in the NGS library, however, additional quantitative analysis as quantitative PCR (qPCR) or digital PCR (dPCR) is required. Furthermore, number of sequences in a sequence library does not always reflect the original copy number of a target gene because of biases caused by PCR amplification, making it difficult to convert the proportion of particular sequences in the NGS library to the copy number using the mass of input DNA. To address this issue, we applied stochastic labeling approach with random-tag sequences and developed a NGS-based quantification protocol, which enables simultaneous sequencing and quantification of the targeted DNA. This quantitative sequencing (qSeq) is initiated from single-primer extension (SPE) using a primer with random tag adjacent to the 5' end of target-specific sequence. During SPE, each DNA molecule is stochastically labeled with the random tag. Subsequently, first-round PCR is conducted, specifically targeting the SPE product, followed by second-round PCR to index for NGS. The number of random tags is only determined during the SPE step and is therefore not affected by the two rounds of PCR that may introduce amplification biases. In the case of 16S rRNA genes, after NGS sequencing and taxonomic classification, the absolute number of target phylotypes 16S rRNA gene can be estimated by Poisson statistics by counting random tags incorporated at the end of sequence. To test the feasibility of this approach, the 16S rRNA gene of Sulfolobus tokodaii was subjected to qSeq, which resulted in accurate quantification of 5.0 × 103 to 5.0 × 104 copies of the 16S rRNA gene. Furthermore, qSeq was applied to mock microbial communities and environmental samples, and the results were comparable to those obtained using digital PCR and

  15. Computational Prediction of miRNA Genes from Small RNA Sequencing Data

    PubMed Central

    Kang, Wenjing; Friedländer, Marc R.

    2015-01-01

    Next-generation sequencing now for the first time allows researchers to gage the depth and variation of entire transcriptomes. However, now as rare transcripts can be detected that are present in cells at single copies, more advanced computational tools are needed to accurately annotate and profile them. microRNAs (miRNAs) are 22 nucleotide small RNAs (sRNAs) that post-transcriptionally reduce the output of protein coding genes. They have established roles in numerous biological processes, including cancers and other diseases. During miRNA biogenesis, the sRNAs are sequentially cleaved from precursor molecules that have a characteristic hairpin RNA structure. The vast majority of new miRNA genes that are discovered are mined from small RNA sequencing (sRNA-seq), which can detect more than a billion RNAs in a single run. However, given that many of the detected RNAs are degradation products from all types of transcripts, the accurate identification of miRNAs remain a non-trivial computational problem. Here, we review the tools available to predict animal miRNAs from sRNA sequencing data. We present tools for generalist and specialist use cases, including prediction from massively pooled data or in species without reference genome. We also present wet-lab methods used to validate predicted miRNAs, and approaches to computationally benchmark prediction accuracy. For each tool, we reference validation experiments and benchmarking efforts. Last, we discuss the future of the field. PMID:25674563

  16. Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys

    PubMed Central

    Berry, David; Gutierrez, Tony

    2017-01-01

    Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira, and Thalassolituus, as well as the Alphaproteobacterial genus Thalassospira. Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys. PMID:28567035

  17. Evaluating the Detection of Hydrocarbon-Degrading Bacteria in 16S rRNA Gene Sequencing Surveys.

    PubMed

    Berry, David; Gutierrez, Tony

    2017-01-01

    Hydrocarbonoclastic bacteria (HCB) play a key role in the biodegradation of oil hydrocarbons in marine and other environments. A small number of taxa have been identified as obligate HCB, notably the Gammaproteobacterial genera Alcanivorax, Cycloclasticus, Marinobacter, Neptumonas, Oleiphilus, Oleispira , and Thalassolituus , as well as the Alphaproteobacterial genus Thalassospira . Detection of HCB in amplicon-based sequencing surveys relies on high coverage by PCR primers and accurate taxonomic classification. In this study, we performed a phylogenetic analysis to identify 16S rRNA gene sequence regions that represent the breadth of sequence diversity within these taxa. Using validated sequences, we evaluated 449 universal 16S rRNA gene-targeted bacterial PCR primer pairs for their coverage of these taxa. The results of this analysis provide a practical framework for selection of suitable primer sets for optimal detection of HCB in sequencing surveys.

  18. An adaptive, object oriented strategy for base calling in DNA sequence analysis.

    PubMed Central

    Giddings, M C; Brumley, R L; Haker, M; Smith, L M

    1993-01-01

    An algorithm has been developed for the determination of nucleotide sequence from data produced in fluorescence-based automated DNA sequencing instruments employing the four-color strategy. This algorithm takes advantage of object oriented programming techniques for modularity and extensibility. The algorithm is adaptive in that data sets from a wide variety of instruments and sequencing conditions can be used with good results. Confidence values are provided on the base calls as an estimate of accuracy. The algorithm iteratively employs confidence determinations from several different modules, each of which examines a different feature of the data for accurate peak identification. Modules within this system can be added or removed for increased performance or for application to a different task. In comparisons with commercial software, the algorithm performed well. Images PMID:8233787

  19. Impact of Next Generation Sequencing Techniques in Food Microbiology

    PubMed Central

    Mayo, Baltasar; Rachid, Caio T. C. C; Alegría, Ángel; Leite, Analy M. O; Peixoto, Raquel S; Delgado, Susana

    2014-01-01

    Understanding the Maxam-Gilbert and Sanger sequencing as the first generation, in recent years there has been an explosion of newly-developed sequencing strategies, which are usually referred to as next generation sequencing (NGS) techniques. NGS techniques have high-throughputs and produce thousands or even millions of sequences at the same time. These sequences allow for the accurate identification of microbial taxa, including uncultivable organisms and those present in small numbers. In specific applications, NGS provides a complete inventory of all microbial operons and genes present or being expressed under different study conditions. NGS techniques are revolutionizing the field of microbial ecology and have recently been used to examine several food ecosystems. After a short introduction to the most common NGS systems and platforms, this review addresses how NGS techniques have been employed in the study of food microbiota and food fermentations, and discusses their limits and perspectives. The most important findings are reviewed, including those made in the study of the microbiota of milk, fermented dairy products, and plant-, meat- and fish-derived fermented foods. The knowledge that can be gained on microbial diversity, population structure and population dynamics via the use of these technologies could be vital in improving the monitoring and manipulation of foods and fermented food products. They should also improve their safety. PMID:25132799

  20. Learning Quantitative Sequence-Function Relationships from Massively Parallel Experiments

    NASA Astrophysics Data System (ADS)

    Atwal, Gurinder S.; Kinney, Justin B.

    2016-03-01

    A fundamental aspect of biological information processing is the ubiquity of sequence-function relationships—functions that map the sequence of DNA, RNA, or protein to a biochemically relevant activity. Most sequence-function relationships in biology are quantitative, but only recently have experimental techniques for effectively measuring these relationships been developed. The advent of such "massively parallel" experiments presents an exciting opportunity for the concepts and methods of statistical physics to inform the study of biological systems. After reviewing these recent experimental advances, we focus on the problem of how to infer parametric models of sequence-function relationships from the data produced by these experiments. Specifically, we retrace and extend recent theoretical work showing that inference based on mutual information, not the standard likelihood-based approach, is often necessary for accurately learning the parameters of these models. Closely connected with this result is the emergence of "diffeomorphic modes"—directions in parameter space that are far less constrained by data than likelihood-based inference would suggest. Analogous to Goldstone modes in physics, diffeomorphic modes arise from an arbitrarily broken symmetry of the inference problem. An analytically tractable model of a massively parallel experiment is then described, providing an explicit demonstration of these fundamental aspects of statistical inference. This paper concludes with an outlook on the theoretical and computational challenges currently facing studies of quantitative sequence-function relationships.