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      Practical Guidelines for the Comprehensive Analysis of ChIP-seq Data

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          Abstract

          Mapping the chromosomal locations of transcription factors, nucleosomes, histone modifications, chromatin remodeling enzymes, chaperones, and polymerases is one of the key tasks of modern biology, as evidenced by the Encyclopedia of DNA Elements (ENCODE) Project. To this end, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) is the standard methodology. Mapping such protein-DNA interactions in vivo using ChIP-seq presents multiple challenges not only in sample preparation and sequencing but also for computational analysis. Here, we present step-by-step guidelines for the computational analysis of ChIP-seq data. We address all the major steps in the analysis of ChIP-seq data: sequencing depth selection, quality checking, mapping, data normalization, assessment of reproducibility, peak calling, differential binding analysis, controlling the false discovery rate, peak annotation, visualization, and motif analysis. At each step in our guidelines we discuss some of the software tools most frequently used. We also highlight the challenges and problems associated with each step in ChIP-seq data analysis. We present a concise workflow for the analysis of ChIP-seq data in Figure 1 that complements and expands on the recommendations of the ENCODE and modENCODE projects. Each step in the workflow is described in detail in the following sections.

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          Most cited references 65

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          Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences

          Increased reliance on computational approaches in the life sciences has revealed grave concerns about how accessible and reproducible computation-reliant results truly are. Galaxy http://usegalaxy.org, an open web-based platform for genomic research, addresses these problems. Galaxy automatically tracks and manages data provenance and provides support for capturing the context and intent of computational methods. Galaxy Pages are interactive, web-based documents that provide users with a medium to communicate a complete computational analysis.
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            Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs

            RNA-Seq provides an unbiased way to study a transcriptome, including both coding and non-coding genes. To date, most RNA-Seq studies have critically depended on existing annotations, and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We apply it to mouse embryonic stem cells, neuronal precursor cells, and lung fibroblasts to accurately reconstruct the full-length gene structures for the vast majority of known expressed genes. We identify substantial variation in protein-coding genes, including thousands of novel 5′-start sites, 3′-ends, and internal coding exons. We then determine the gene structures of over a thousand lincRNA and antisense loci. Our results open the way to direct experimental manipulation of thousands of non-coding RNAs, and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes.
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              A clustering approach for identification of enriched domains from histone modification ChIP-Seq data.

              Chromatin states are the key to gene regulation and cell identity. Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-Seq) is increasingly being used to map epigenetic states across genomes of diverse species. Chromatin modification profiles are frequently noisy and diffuse, spanning regions ranging from several nucleosomes to large domains of multiple genes. Much of the early work on the identification of ChIP-enriched regions for ChIP-Seq data has focused on identifying localized regions, such as transcription factor binding sites. Bioinformatic tools to identify diffuse domains of ChIP-enriched regions have been lacking. Based on the biological observation that histone modifications tend to cluster to form domains, we present a method that identifies spatial clusters of signals unlikely to appear by chance. This method pools together enrichment information from neighboring nucleosomes to increase sensitivity and specificity. By using genomic-scale analysis, as well as the examination of loci with validated epigenetic states, we demonstrate that this method outperforms existing methods in the identification of ChIP-enriched signals for histone modification profiles. We demonstrate the application of this unbiased method in important issues in ChIP-Seq data analysis, such as data normalization for quantitative comparison of levels of epigenetic modifications across cell types and growth conditions. http://home.gwu.edu/ approximately wpeng/Software.htm. Supplementary data are available at Bioinformatics online.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Comput Biol
                PLoS Comput. Biol
                plos
                ploscomp
                PLoS Computational Biology
                Public Library of Science (San Francisco, USA )
                1553-734X
                1553-7358
                November 2013
                November 2013
                14 November 2013
                : 9
                : 11
                Affiliations
                [1 ]Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia
                [2 ]Department of Biometry and Bioinformatics, Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland
                [3 ]Department of Statistics, Beadle Center, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America
                [4 ]Inserm U981, Cancer Institute Gustave Roussy, Villejuif, France
                [5 ]Department of Statistics, Penn State University, University Park, Pennsylvania, United States of America
                [6 ]Department of Biochemistry, University at Buffalo, Buffalo, New York, United States of America
                [7 ]Department of Biomedical Informatics, The Ohio State University, Columbus, Ohio, United States of America
                Whitehead Institute, United States of America
                Author notes

                ¶ All authors contributed equally to this manuscript.

                The authors have declared that no competing interests exist.

                Article
                PCOMPBIOL-D-13-00917
                10.1371/journal.pcbi.1003326
                3828144
                24244136

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 8
                Funding
                TLB is funded by National Institutes of Health grant R0-1 RR021692-05. PM was supported by the EU Marie Curie Initial Training Network SYSFLO (agreement number 237909). QL is partially supported by National Institutes of Health grants 1UL1 RR033184-01 and R01 GM109453. IL gratefully acknowledges support from UNL IANR. The funders had no role in the preparation of the manuscript.
                Categories
                Education

                Quantitative & Systems biology

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