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  • 1
    Publication Date: 2015-09-19
    Description: Structural variations (SVs) play a crucial role in genetic diversity. However, the alignments of reads near/across SVs are made inaccurate by the presence of polymorphisms. BatAlign is an algorithm that integrated two strategies called ‘Reverse-Alignment’ and ‘Deep-Scan’ to improve the accuracy of read-alignment. In our experiments, BatAlign was able to obtain the highest F-measures in read-alignments on mismatch-aberrant, indel-aberrant, concordantly/discordantly paired and SV-spanning data sets. On real data, the alignments of BatAlign were able to recover 4.3% more PCR-validated SVs with 73.3% less callings. These suggest BatAlign to be effective in detecting SVs and other polymorphic-variants accurately using high-throughput data. BatAlign is publicly available at https://goo.gl/a6phxB .
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 2
    Publication Date: 2016-03-01
    Description: The adaptive immune system includes populations of B and T cells capable of binding foreign epitopes via antigen specific receptors, called immunoglobulin (IG) for B cells and the T cell receptor (TCR) for T cells. In order to provide protection from a wide range of pathogens, these cells display highly diverse repertoires of IGs and TCRs. This is achieved through combinatorial rearrangement of multiple gene segments in addition, for B cells, to somatic hypermutation. Deep sequencing technologies have revolutionized analysis of the diversity of these repertoires; however, accurate TCR/IG diversity profiling requires specialist bioinformatics tools. Here we present LymAnalzyer, a software package that significantly improves the completeness and accuracy of TCR/IG profiling from deep sequence data and includes procedures to identify novel alleles of gene segments. On real and simulated data sets LymAnalyzer produces highly accurate and complete results. Although, to date we have applied it to TCR/IG data from human and mouse, it can be applied to data from any species for which an appropriate database of reference genes is available. Implemented in Java, it includes both a command line version and a graphical user interface and is freely available at https://sourceforge.net/projects/lymanalyzer/ .
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 3
    Publication Date: 2012-05-13
    Description: Quantitative analyses of next-generation sequencing (NGS) data, such as the detection of copy number variations (CNVs), remain challenging. Current methods detect CNVs as changes in the depth of coverage along chromosomes. Technological or genomic variations in the depth of coverage thus lead to a high false discovery rate (FDR), even upon correction for GC content. In the context of association studies between CNVs and disease, a high FDR means many false CNVs, thereby decreasing the discovery power of the study after correction for multiple testing. We propose ‘Copy Number estimation by a Mixture Of PoissonS’ (cn.MOPS), a data processing pipeline for CNV detection in NGS data. In contrast to previous approaches, cn.MOPS incorporates modeling of depths of coverage across samples at each genomic position. Therefore, cn.MOPS is not affected by read count variations along chromosomes. Using a Bayesian approach, cn.MOPS decomposes variations in the depth of coverage across samples into integer copy numbers and noise by means of its mixture components and Poisson distributions, respectively. The noise estimate allows for reducing the FDR by filtering out detections having high noise that are likely to be false detections. We compared cn.MOPS with the five most popular methods for CNV detection in NGS data using four benchmark datasets: (i) simulated data, (ii) NGS data from a male HapMap individual with implanted CNVs from the X chromosome, (iii) data from HapMap individuals with known CNVs, (iv) high coverage data from the 1000 Genomes Project. cn.MOPS outperformed its five competitors in terms of precision (1–FDR) and recall for both gains and losses in all benchmark data sets. The software cn.MOPS is publicly available as an R package at http://www.bioinf.jku.at/software/cnmops/ and at Bioconductor.
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 4
    Publication Date: 2012-05-23
    Description: GC content bias describes the dependence between fragment count (read coverage) and GC content found in Illumina sequencing data. This bias can dominate the signal of interest for analyses that focus on measuring fragment abundance within a genome, such as copy number estimation (DNA-seq). The bias is not consistent between samples; and there is no consensus as to the best methods to remove it in a single sample. We analyze regularities in the GC bias patterns, and find a compact description for this unimodal curve family. It is the GC content of the full DNA fragment, not only the sequenced read, that most influences fragment count. This GC effect is unimodal: both GC-rich fragments and AT-rich fragments are underrepresented in the sequencing results. This empirical evidence strengthens the hypothesis that PCR is the most important cause of the GC bias. We propose a model that produces predictions at the base pair level, allowing strand-specific GC-effect correction regardless of the downstream smoothing or binning. These GC modeling considerations can inform other high-throughput sequencing analyses such as ChIP-seq and RNA-seq.
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 5
    Publication Date: 2012-03-29
    Description: Recent advances in sequencing technology have enabled the rapid generation of billions of bases at relatively low cost. A crucial first step in many sequencing applications is to map those reads to a reference genome. However, when the reference genome is large, finding accurate mappings poses a significant computational challenge due to the sheer amount of reads, and because many reads map to the reference sequence approximately but not exactly. We introduce Hobbes, a new gram-based program for aligning short reads, supporting Hamming and edit distance. Hobbes implements two novel techniques, which yield substantial performance improvements: an optimized gram-selection procedure for reads, and a cache-efficient filter for pruning candidate mappings. We systematically tested the performance of Hobbes on both real and simulated data with read lengths varying from 35 to 100 bp, and compared its performance with several state-of-the-art read-mapping programs, including Bowtie, BWA, mrsFast and RazerS. Hobbes is faster than all other read mapping programs we have tested while maintaining high mapping quality. Hobbes is about five times faster than Bowtie and about 2–10 times faster than BWA, depending on read length and error rate, when asked to find all mapping locations of a read in the human genome within a given Hamming or edit distance, respectively. Hobbes supports the SAM output format and is publicly available at http://hobbes.ics.uci.edu .
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 6
    Publication Date: 2013-09-06
    Description: MiST is a novel approach to variant calling from deep sequencing data, using the inverted mapping approach developed for Geoseq. Reads that can map to a targeted exonic region are identified using exact matches to tiles from the region. The reads are then aligned to the targets to discover variants. MiST carefully handles paralogous reads that map ambiguously to the genome and clonal reads arising from PCR bias, which are the two major sources of errors in variant calling. The reduced computational complexity of mapping selected reads to targeted regions of the genome improves speed, specificity and sensitivity of variant detection. Compared with variant calls from the GATK platform, MiST showed better concordance with SNPs from dbSNP and genotypes determined by an exonic-SNP array. Variant calls made only by MiST confirm at a high rate (〉90%) by Sanger sequencing. Thus, MiST is a valuable alternative tool to analyse variants in deep sequencing data.
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
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    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 7
    Publication Date: 2012-12-14
    Description: We present Quip, a lossless compression algorithm for next-generation sequencing data in the FASTQ and SAM/BAM formats. In addition to implementing reference-based compression, we have developed, to our knowledge, the first assembly-based compressor, using a novel de novo assembly algorithm. A probabilistic data structure is used to dramatically reduce the memory required by traditional de Bruijn graph assemblers, allowing millions of reads to be assembled very efficiently. Read sequences are then stored as positions within the assembled contigs. This is combined with statistical compression of read identifiers, quality scores, alignment information and sequences, effectively collapsing very large data sets to 〈15% of their original size with no loss of information. Availability: Quip is freely available under the 3-clause BSD license from http://cs.washington.edu/homes/dcjones/quip .
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
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    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 8
    Publication Date: 2013-05-29
    Description: Sequencing of RNAs (RNA-Seq) has revolutionized the field of transcriptomics, but the reads obtained often contain errors. Read error correction can have a large impact on our ability to accurately assemble transcripts. This is especially true for de novo transcriptome analysis, where a reference genome is not available. Current read error correction methods, developed for DNA sequence data, cannot handle the overlapping effects of non-uniform abundance, polymorphisms and alternative splicing. Here we present SEquencing Error CorrEction in Rna-seq data (SEECER), a hidden Markov Model (HMM)–based method, which is the first to successfully address these problems. SEECER efficiently learns hundreds of thousands of HMMs and uses these to correct sequencing errors. Using human RNA-Seq data, we show that SEECER greatly improves on previous methods in terms of quality of read alignment to the genome and assembly accuracy. To illustrate the usefulness of SEECER for de novo transcriptome studies, we generated new RNA-Seq data to study the development of the sea cucumber Parastichopus parvimensis . Our corrected assembled transcripts shed new light on two important stages in sea cucumber development. Comparison of the assembled transcripts to known transcripts in other species has also revealed novel transcripts that are unique to sea cucumber, some of which we have experimentally validated. Supporting website: http://sb.cs.cmu.edu/seecer/ .
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
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    Topics: Biology
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  • 9
    Publication Date: 2013-11-21
    Description: Detection of differential expression in RNA-Seq data is currently limited to studies in which two or more sample conditions are known a priori. However, these biological conditions are typically unknown in cohort, cross-sectional and nonrandomized controlled studies such as the HapMap, the ENCODE or the 1000 Genomes project. We present DEXUS for detecting differential expression in RNA-Seq data for which the sample conditions are unknown. DEXUS models read counts as a finite mixture of negative binomial distributions in which each mixture component corresponds to a condition. A transcript is considered differentially expressed if modeling of its read counts requires more than one condition. DEXUS decomposes read count variation into variation due to noise and variation due to differential expression. Evidence of differential expression is measured by the informative/noninformative (I/NI) value, which allows differentially expressed transcripts to be extracted at a desired specificity (significance level) or sensitivity (power). DEXUS performed excellently in identifying differentially expressed transcripts in data with unknown conditions. On 2400 simulated data sets, I/NI value thresholds of 0.025, 0.05 and 0.1 yielded average specificities of 92, 97 and 99% at sensitivities of 76, 61 and 38%, respectively. On real-world data sets, DEXUS was able to detect differentially expressed transcripts related to sex, species, tissue, structural variants or quantitative trait loci. The DEXUS R package is publicly available from Bioconductor and the scripts for all experiments are available at http://www.bioinf.jku.at/software/dexus/ .
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 10
    Publication Date: 2012-09-27
    Description: High-throughput immunoglobulin sequencing promises new insights into the somatic hypermutation and antigen-driven selection processes that underlie B-cell affinity maturation and adaptive immunity. The ability to estimate positive and negative selection from these sequence data has broad applications not only for understanding the immune response to pathogens, but is also critical to determining the role of somatic hypermutation in autoimmunity and B-cell cancers. Here, we develop a statistical framework for Bayesian estimation of Antigen-driven SELectIoN (BASELINe) based on the analysis of somatic mutation patterns. Our approach represents a fundamental advance over previous methods by shifting the problem from one of simply detecting selection to one of quantifying selection. Along with providing a more intuitive means to assess and visualize selection, our approach allows, for the first time, comparative analysis between groups of sequences derived from different germline V(D)J segments. Application of this approach to next-generation sequencing data demonstrates different selection pressures for memory cells of different isotypes. This framework can easily be adapted to analyze other types of DNA mutation patterns resulting from a mutator that displays hot/cold-spots, substitution preference or other intrinsic biases.
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
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    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 11
    Publication Date: 2012-09-27
    Description: Competitive gene set tests are commonly used in molecular pathway analysis to test for enrichment of a particular gene annotation category amongst the differential expression results from a microarray experiment. Existing gene set tests that rely on gene permutation are shown here to be extremely sensitive to inter-gene correlation. Several data sets are analyzed to show that inter-gene correlation is non-ignorable even for experiments on homogeneous cell populations using genetically identical model organisms. A new gene set test procedure (CAMERA) is proposed based on the idea of estimating the inter-gene correlation from the data, and using it to adjust the gene set test statistic. An efficient procedure is developed for estimating the inter-gene correlation and characterizing its precision. CAMERA is shown to control the type I error rate correctly regardless of inter-gene correlations, yet retains excellent power for detecting genuine differential expression. Analysis of breast cancer data shows that CAMERA recovers known relationships between tumor subtypes in very convincing terms. CAMERA can be used to analyze specified sets or as a pathway analysis tool using a database of molecular signatures.
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 12
    Publication Date: 2013-04-14
    Description: With the advent of high-throughput sequencing technologies, the rapid generation and accumulation of large amounts of sequencing data pose an insurmountable demand for efficient algorithms for constructing whole-genome phylogenies. The existing phylogenomic methods all use assembled sequences, which are often not available owing to the difficulty of assembling short-reads; this obstructs phylogenetic investigations on species without a reference genome. In this report, we present co-phylog , an assembly-free phylogenomic approach that creates a ‘micro-alignment’ at each ‘object’ in the sequence using the ‘context’ of the object and calculates pairwise distances before reconstructing the phylogenetic tree based on those distances. We explored the parameters’ usages and the optimal working range of co-phylog , assessed co-phylog using the simulated next-generation sequencing (NGS) data and the real NGS raw data. We also compared co-phylog method with traditional alignment and alignment-free methods and illustrated the advantages and limitations of co-phylog method. In conclusion, we demonstrated that co-phylog is efficient algorithm and that it delivers high resolution and accurate phylogenies using whole-genome unassembled sequencing data, especially in the case of closely related organisms, thereby significantly alleviating the computational burden in the genomic era.
    Keywords: Computational Methods, Massively Parallel (Deep) Sequencing
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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