Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers
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 solut...
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| Veröffentlicht in: | PloS one 2016-07, Vol.11 (7), p.e0160227-e0160227 |
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| creator | Pabinger, Stephan Ernst, Karina Pulverer, Walter Kallmeyer, Rainer Valdes, Ana M Metrustry, Sarah Katic, Denis Nuzzo, Angelo Kriegner, Albert Vierlinger, Klemens Weinhaeusel, Andreas |
| description | 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 available under a GNU General Public License version 3.0 (GPLv3) at https://github.com/tadkeys/tabsat/ and http://demo.platomics.com/. |
| doi_str_mv | 10.1371/journal.pone.0160227 |
| format | Article |
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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 available under a GNU General Public License version 3.0 (GPLv3) at https://github.com/tadkeys/tabsat/ and http://demo.platomics.com/.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0160227</identifier><identifier>PMID: 27467908</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Analysis ; Bioinformatics ; Biology and life sciences ; Bisulfite ; Computer and Information Sciences ; Correlation coefficient ; Correlation coefficients ; CpG islands ; Data processing ; Deoxyribonucleic acid ; DNA ; DNA - chemistry ; DNA Methylation ; DNA sequencing ; Epidemiology ; Epigenetics ; Gene expression ; Gene sequencing ; Genomes ; Graphical user interface ; Humans ; Mathematical analysis ; Medical diagnostic software ; Methylation ; Nucleotide sequence ; Performance assessment ; Physical Sciences ; Polymerase chain reaction ; Polymerase Chain Reaction - methods ; Protocol (computers) ; Quality ; Quality assessment ; Quality control ; Research and analysis methods ; Sequence Analysis, DNA - methods ; Studies ; Sulfites - chemistry ; Technology application ; Workflow</subject><ispartof>PloS one, 2016-07, Vol.11 (7), p.e0160227-e0160227</ispartof><rights>COPYRIGHT 2016 Public Library of Science</rights><rights>2016 Pabinger et al. 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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 available under a GNU General Public License version 3.0 (GPLv3) at https://github.com/tadkeys/tabsat/ and http://demo.platomics.com/.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>27467908</pmid><doi>10.1371/journal.pone.0160227</doi><tpages>e0160227</tpages><oa>free_for_read</oa></addata></record> |
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| language | eng |
| recordid | cdi_plos_journals_1807561749 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS); PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Analysis Bioinformatics Biology and life sciences Bisulfite Computer and Information Sciences Correlation coefficient Correlation coefficients CpG islands Data processing Deoxyribonucleic acid DNA DNA - chemistry DNA Methylation DNA sequencing Epidemiology Epigenetics Gene expression Gene sequencing Genomes Graphical user interface Humans Mathematical analysis Medical diagnostic software Methylation Nucleotide sequence Performance assessment Physical Sciences Polymerase chain reaction Polymerase Chain Reaction - methods Protocol (computers) Quality Quality assessment Quality control Research and analysis methods Sequence Analysis, DNA - methods Studies Sulfites - chemistry Technology application Workflow |
| title | Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-11T19%3A19%3A56IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Analysis%20and%20Visualization%20Tool%20for%20Targeted%20Amplicon%20Bisulfite%20Sequencing%20on%20Ion%20Torrent%20Sequencers&rft.jtitle=PloS%20one&rft.au=Pabinger,%20Stephan&rft.date=2016-07-28&rft.volume=11&rft.issue=7&rft.spage=e0160227&rft.epage=e0160227&rft.pages=e0160227-e0160227&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0160227&rft_dat=%3Cgale_plos_%3EA459396460%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1807561749&rft_id=info:pmid/27467908&rft_galeid=A459396460&rft_doaj_id=oai_doaj_org_article_6a14e6fdfd184156b647c253c494941d&rfr_iscdi=true |