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      Methy-Pipe: An Integrated Bioinformatics Pipeline for Whole Genome Bisulfite Sequencing Data Analysis

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          Abstract

          DNA methylation, one of the most important epigenetic modifications, plays a crucial role in various biological processes. The level of DNA methylation can be measured using whole-genome bisulfite sequencing at single base resolution. However, until now, there is a paucity of publicly available software for carrying out integrated methylation data analysis. In this study, we implemented Methy-Pipe, which not only fulfills the core data analysis requirements (e.g. sequence alignment, differential methylation analysis, etc.) but also provides useful tools for methylation data annotation and visualization. Specifically, it uses Burrow-Wheeler Transform (BWT) algorithm to directly align bisulfite sequencing reads to a reference genome and implements a novel sliding window based approach with statistical methods for the identification of differentially methylated regions (DMRs). The capability of processing data parallelly allows it to outperform a number of other bisulfite alignment software packages. To demonstrate its utility and performance, we applied it to both real and simulated bisulfite sequencing datasets. The results indicate that Methy-Pipe can accurately estimate methylation densities, identify DMRs and provide a variety of utility programs for downstream methylation data analysis. In summary, Methy-Pipe is a useful pipeline that can process whole genome bisulfite sequencing data in an efficient, accurate, and user-friendly manner. Software and test dataset are available at http://sunlab.lihs.cuhk.edu.hk/methy-pipe/.

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          Most cited references8

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          Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.

          Cytosine DNA methylation is important in regulating gene expression and in silencing transposons and other repetitive sequences. Recent genomic studies in Arabidopsis thaliana have revealed that many endogenous genes are methylated either within their promoters or within their transcribed regions, and that gene methylation is highly correlated with transcription levels. However, plants have different types of methylation controlled by different genetic pathways, and detailed information on the methylation status of each cytosine in any given genome is lacking. To this end, we generated a map at single-base-pair resolution of methylated cytosines for Arabidopsis, by combining bisulphite treatment of genomic DNA with ultra-high-throughput sequencing using the Illumina 1G Genome Analyser and Solexa sequencing technology. This approach, termed BS-Seq, unlike previous microarray-based methods, allows one to sensitively measure cytosine methylation on a genome-wide scale within specific sequence contexts. Here we describe methylation on previously inaccessible components of the genome and analyse the DNA methylation sequence composition and distribution. We also describe the effect of various DNA methylation mutants on genome-wide methylation patterns, and demonstrate that our newly developed library construction and computational methods can be applied to large genomes such as that of mouse.
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            BS Seeker: precise mapping for bisulfite sequencing

            Background Bisulfite sequencing using next generation sequencers yields genome-wide measurements of DNA methylation at single nucleotide resolution. Traditional aligners are not designed for mapping bisulfite-treated reads, where the unmethylated Cs are converted to Ts. We have developed BS Seeker, an approach that converts the genome to a three-letter alphabet and uses Bowtie to align bisulfite-treated reads to a reference genome. It uses sequence tags to reduce mapping ambiguity. Post-processing of the alignments removes non-unique and low-quality mappings. Results We tested our aligner on synthetic data, a bisulfite-converted Arabidopsis library, and human libraries generated from two different experimental protocols. We evaluated the performance of our approach and compared it to other bisulfite aligners. The results demonstrate that among the aligners tested, BS Seeker is more versatile and faster. When mapping to the human genome, BS Seeker generates alignments significantly faster than RMAP and BSMAP. Furthermore, BS Seeker is the only alignment tool that can explicitly account for tags which are generated by certain library construction protocols. Conclusions BS Seeker provides fast and accurate mapping of bisulfite-converted reads. It can work with BS reads generated from the two different experimental protocols, and is able to efficiently map reads to large mammalian genomes. The Python program is freely available at http://pellegrini.mcdb.ucla.edu/BS_Seeker/BS_Seeker.html.
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              DNA methylation and the regulation of gene transcription.

              The regulation of gene transcription is not simply dependent on the presence or absence of DNA-binding transcription factors that turn genes on or off, but also involves processes determining the ability of transcription factors to gain access to and bind their target DNA. Methylation of DNA cytosine bases leads to the inaccessibility of DNA regulatory elements to their transcription factors by a number of mechanisms. Our understanding of DNA methylation has advanced rapidly in recent years with the identification of an increasingly large number of novel proteins involved in this process. These include methylcytosine-binding proteins as well as additional members of the DNA methyltransferase family. The creation of mice with targeted deletions in a number of genes involved in DNA methylation has further elucidated the functions of many of these proteins. The characterization of complexes that contain proteins known to be involved in DNA methylation has led to the identification of additional proteins, especially those involved in histone deacetylation, indicating that DNA methylation and histone deacetylation very likely act in a synergistic fashion to regulate gene transcription. Finally, the implication of DNA methylation in tumorigenesis and the realization that some congenital diseases are caused by deficiency of proteins involved in DNA methylation has confirmed the importance of this process in regulating gene expression.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                19 June 2014
                : 9
                : 6
                : e100360
                Affiliations
                [1 ]Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China
                [2 ]Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China
                [3 ]Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China
                Wayne State University, United States of America
                Author notes

                Competing Interests: Peiyong Jiang, Fiona M.F. Lun, K.C. Allen Chan, Rossa W.K. Chiu and Y.M. Dennis Lo have filed a United States patent application 13/842,209 “Noninvasive determination of methylome of fetus or tumor from plasma”. Y.M. Dennis Lo and Rossa W.K. Chiu are consultants to, receive research support from, and hold equities in Sequenom. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

                Conceived and designed the experiments: PJ KS KCAC RWKC YMDL HS. Performed the experiments: FMFL. Analyzed the data: PJ KS AMG HS. Wrote the paper: PJ KS HW HS.

                Article
                PONE-D-14-10607
                10.1371/journal.pone.0100360
                4063866
                24945300
                c8771186-e967-404c-bf54-e8501ca7f467
                Copyright @ 2014

                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.

                History
                : 8 March 2014
                : 23 May 2014
                Page count
                Pages: 11
                Funding
                This work is supported by the University Grants Committee of the Government of the Hong Kong Special Administrative Region, China, under the Areas of Excellence Scheme (AoE/M-04/06). The Hong Kong Research Grants Council General Research Fund (CUHK473713). Y.M.D. Lo was supported by an Endowed Chair from the Li Ka Shing Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Biochemistry
                DNA
                DNA modification
                DNA methylation
                Computational Biology
                Genetics
                Epigenetics
                Research and Analysis Methods
                Database and Informatics Methods
                Bioinformatics

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                Uncategorized

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