title: Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers.
abstract: 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/.
authors.affiliation: Health & Environment Department, Molecular Diagnostics, AIT-Austrian Institute of Technology, Vienna, Austria.
authors.forename: Stephan
authors.initials: S
authors.lastname: Pabinger
authors.affiliation: Health & Environment Department, Molecular Diagnostics, AIT-Austrian Institute of Technology, Vienna, Austria.
authors.forename: Karina
authors.initials: K
authors.lastname: Ernst
authors.affiliation: Health & Environment Department, Molecular Diagnostics, AIT-Austrian Institute of Technology, Vienna, Austria.
authors.forename: Walter
authors.initials: W
authors.lastname: Pulverer
authors.affiliation: Health & Environment Department, Molecular Diagnostics, AIT-Austrian Institute of Technology, Vienna, Austria.
authors.forename: Rainer
authors.initials: R
authors.lastname: Kallmeyer
authors.affiliation: The Department of Twin Research & Genetic Epidemiology, King's College London, St Thomas' Campus, London, United Kingdom.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Valdes
authors.affiliation: The Department of Twin Research & Genetic Epidemiology, King's College London, St Thomas' Campus, London, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Metrustry
authors.affiliation: Platomics GmbH, Vienna, Austria.
authors.forename: Denis
authors.initials: D
authors.lastname: Katic
authors.affiliation: Platomics GmbH, Vienna, Austria.
authors.forename: Angelo
authors.initials: A
authors.lastname: Nuzzo
authors.affiliation: Platomics GmbH, Vienna, Austria.
authors.forename: Albert
authors.initials: A
authors.lastname: Kriegner
authors.affiliation: Health & Environment Department, Molecular Diagnostics, AIT-Austrian Institute of Technology, Vienna, Austria.
authors.forename: Klemens
authors.initials: K
authors.lastname: Vierlinger
authors.affiliation: Health & Environment Department, Molecular Diagnostics, AIT-Austrian Institute of Technology, Vienna, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Weinhaeusel
mesh_terms: D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000737:chemistry*
chemical_list: D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite