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  • 1
    Publication Date: 2012-07-22
    Description: Establishing the functional roles of genetic variants remains a significant challenge in the post-genomic era. Here, we present a method, allele-specific alternative mRNA processing (ASARP), to identify genetically influenced mRNA processing events using transcriptome sequencing (RNA-Seq) data. The method examines RNA-Seq data at both single-nucleotide and whole-gene/isoform levels to identify allele-specific expression (ASE) and existence of allele-specific regulation of mRNA processing. We applied the methods to data obtained from the human glioblastoma cell line U87MG and primary breast cancer tissues and found that 26–45% of all genes with sufficient read coverage demonstrated ASE, with significant overlap between the two cell types. Our methods predicted potential mechanisms underlying ASE due to regulations affecting either whole-gene-level expression or alternative mRNA processing, including alternative splicing, alternative polyadenylation and alternative transcriptional initiation. Allele-specific alternative splicing and alternative polyadenylation may explain ASE in hundreds of genes in each cell type. Reporter studies following these predictions identified the causal single nucleotide variants (SNVs) for several allele-specific alternative splicing events. Finally, many genes identified in our study were also reported as disease/phenotype-associated genes in genome-wide association studies. Future applications of our approach may provide ample insights for a better understanding of the genetic basis of gene regulation underlying phenotypic diversity and disease mechanisms.
    Keywords: Massively Parallel (Deep) Sequencing, Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 2
    Publication Date: 2012-09-13
    Description: Accurate estimation of expression levels from RNA-Seq data entails precise mapping of the sequence reads to a reference genome. Because the standard reference genome contains only one allele at any given locus, reads overlapping polymorphic loci that carry a non-reference allele are at least one mismatch away from the reference and, hence, are less likely to be mapped. This bias in read mapping leads to inaccurate estimates of allele-specific expression (ASE). To address this read-mapping bias, we propose the construction of an enhanced reference genome that includes the alternative alleles at known polymorphic loci. We show that mapping to this enhanced reference reduced the read-mapping biases, leading to more reliable estimates of ASE. Experiments on simulated data show that the proposed strategy reduced the number of loci with mapping bias by ≥63% when compared with a previous approach that relies on masking the polymorphic loci and by ≥18% when compared with the standard approach that uses an unaltered reference. When we applied our strategy to actual RNA-Seq data, we found that it mapped up to 15% more reads than the previous approaches and identified many seemingly incorrect inferences made by them.
    Keywords: Massively Parallel (Deep) Sequencing, Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 3
    Publication Date: 2013-12-07
    Description: The emergence of massively parallel sequencing technology has revolutionized microbial profiling, allowing the unprecedented comparison of microbial diversity across time and space in a wide range of host-associated and environmental ecosystems. Although the high-throughput nature of such methods enables the detection of low-frequency bacteria, these advances come at the cost of sequencing read length, limiting the phylogenetic resolution possible by current methods. Here, we present a generic approach for integrating short reads from large genomic regions, thus enabling phylogenetic resolution far exceeding current methods. The approach is based on a mapping to a statistical model that is later solved as a constrained optimization problem. We demonstrate the utility of this method by analyzing human saliva and Drosophila samples, using Illumina single-end sequencing of a 750 bp amplicon of the 16S rRNA gene. Phylogenetic resolution is significantly extended while reducing the number of falsely detected bacteria, as compared with standard single-region Roche 454 Pyrosequencing. Our approach can be seamlessly applied to simultaneous sequencing of multiple genes providing a higher resolution view of the composition and activity of complex microbial communities.
    Keywords: Massively Parallel (Deep) Sequencing, Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 4
    Publication Date: 2014-02-28
    Description: Accurate mapping of spliced RNA-Seq reads to genomic DNA has been known as a challenging problem. Despite significant efforts invested in developing efficient algorithms, with the human genome as a primary focus, the best solution is still not known. A recently introduced tool, TrueSight, has demonstrated better performance compared with earlier developed algorithms such as TopHat and MapSplice. To improve detection of splice junctions, TrueSight uses information on statistical patterns of nucleotide ordering in intronic and exonic DNA. This line of research led to yet another new algorithm, UnSplicer, designed for eukaryotic species with compact genomes where functional alternative splicing is likely to be dominated by splicing noise. Genome-specific parameters of the new algorithm are generated by GeneMark-ES, an ab initio gene prediction algorithm based on unsupervised training. UnSplicer shares several components with TrueSight; the difference lies in the training strategy and the classification algorithm. We tested UnSplicer on RNA-Seq data sets of Arabidopsis thaliana , Caenorhabditis elegans , Cryptococcus neoformans and Drosophila melanogaster . We have shown that splice junctions inferred by UnSplicer are in better agreement with knowledge accumulated on these well-studied genomes than predictions made by earlier developed tools.
    Keywords: Massively Parallel (Deep) Sequencing, Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 5
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    Oxford University Press
    Publication Date: 2012-09-27
    Description: Genome-wide binding data from transcription factor ChIP-seq experiments is the best source of information for inferring the relative DNA-binding affinity of these proteins in vivo . However, standard motif enrichment analysis and motif discovery approaches sometimes fail to correctly identify the binding motif for the ChIP-ed factor. To overcome this problem, we propose ‘central motif enrichment analysis’ (CMEA), which is based on the observation that the positional distribution of binding sites matching the direct-binding motif tends to be unimodal, well centered and maximal in the precise center of the ChIP-seq peak regions. We describe a novel visualization and statistical analysis tool—CentriMo—that identifies the region of maximum central enrichment in a set of ChIP-seq peak regions and displays the positional distributions of predicted sites. Using CentriMo for motif enrichment analysis, we provide evidence that one transcription factor (Nanog) has different binding affinity in vivo than in vitro , that another binds DNA cooperatively (E2f1), and confirm the in vivo affinity of NFIC, rescuing a difficult ChIP-seq data set. In another data set, CentriMo strongly suggests that there is no evidence of direct DNA binding by the ChIP-ed factor (Smad1). CentriMo is now part of the MEME Suite software package available at http://meme.nbcr.net . All data and output files presented here are available at: http://research.imb.uq.edu.au/t.bailey/sd/Bailey2011a .
    Keywords: Massively Parallel (Deep) Sequencing, Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 6
    Publication Date: 2013-04-23
    Description: The metagenomic paradigm allows for an understanding of the metabolic and functional potential of microbes in a community via a study of their proteins. The substrate for protein identification is either the set of individual nucleotide reads generated from metagenomic samples or the set of contig sequences produced by assembling these reads. However, a read-based strategy using reads generated by next-generation sequencing (NGS) technologies, results in an overwhelming majority of partial-length protein predictions. A nucleotide assembly-based strategy does not fare much better, as metagenomic assemblies are typically fragmented and also leave a large fraction of reads unassembled. Here, we present a method for reconstructing complete protein sequences directly from NGS metagenomic data. Our framework is based on a novel short peptide assembler (SPA) that assembles protein sequences from their constituent peptide fragments identified on short reads. The SPA algorithm is based on informed traversals of a de Bruijn graph, defined on an amino acid alphabet, to identify probable paths that correspond to proteins. Using large simulated and real metagenomic data sets, we show that our method outperforms the alternate approach of identifying genes on nucleotide sequence assemblies and generates longer protein sequences that can be more effectively analysed.
    Keywords: Massively Parallel (Deep) Sequencing, Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 7
    Publication Date: 2013-04-14
    Description: High-throughput sequencing is becoming a popular research tool but carries with it considerable costs in terms of computation time, data storage and bandwidth. Meanwhile, some research applications focusing on individual genes or pathways do not necessitate processing of a full sequencing dataset. Thus, it is desirable to partition a large dataset into smaller, manageable, but relevant pieces. We present a toolkit for partitioning raw sequencing data that includes a method for extracting reads that are likely to map onto pre-defined regions of interest. We show the method can be used to extract information about genes of interest from DNA or RNA sequencing samples in a fraction of the time and disk space required to process and store a full dataset. We report speedup factors between 2.6 and 96, depending on settings and samples used. The software is available at http://www.sourceforge.net/projects/triagetools/ .
    Keywords: Massively Parallel (Deep) Sequencing, Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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  • 8
    Publication Date: 2014-01-28
    Description: As next-generation sequencing continues to have an expanding presence in the clinic, the identification of the most cost-effective and robust strategy for identifying copy number changes and translocations in tumor genomes is needed. We hypothesized that performing shallow whole genome sequencing (WGS) of 900–1000-bp inserts (long insert WGS, LI-WGS) improves our ability to detect these events, compared with shallow WGS of 300–400-bp inserts. A priori analyses show that LI-WGS requires less sequencing compared with short insert WGS to achieve a target physical coverage, and that LI-WGS requires less sequence coverage to detect a heterozygous event with a power of 0.99. We thus developed an LI-WGS library preparation protocol based off of Illumina’s WGS library preparation protocol and illustrate the feasibility of performing LI-WGS. We additionally applied LI-WGS to three separate tumor/normal DNA pairs collected from patients diagnosed with different cancers to demonstrate our application of LI-WGS on actual patient samples for identification of somatic copy number alterations and translocations. With the evolution of sequencing technologies and bioinformatics analyses, we show that modifications to current approaches may improve our ability to interrogate cancer genomes.
    Keywords: Massively Parallel (Deep) Sequencing, Genomics
    Print ISSN: 0305-1048
    Electronic ISSN: 1362-4962
    Topics: Biology
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