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/.
Highlights
DNA methylation is one of the most important epigenetic modifications of the eukaryotic genome and plays essential roles in several biological processes, such as alternative splicing [1], regulation of temporal and spatial gene expression [2,3], as well as genome stabilization [4]
DNA is treated with bisulfite, which changes the DNA sequence depending on the methylation status of individual cytosine residues
We have developed TABSAT, a tool for the analysis of targeted bisulfite sequencing data generated on Ion Torrent systems
Summary
DNA methylation is one of the most important epigenetic modifications of the eukaryotic genome and plays essential roles in several biological processes, such as alternative splicing [1], regulation of temporal and spatial gene expression [2,3], as well as genome stabilization [4]. We have developed TABSAT, a tool for the analysis of targeted bisulfite sequencing data generated on Ion Torrent systems Bismark is a software package containing routines to map reads to a reference genome (using Bowtie [25], or alternatively Bowtie 2 [26]) and determine their methylation state It provides an attractive combination of processing speed, genomic coverage, and quantitative accuracy and is one of the most widely used tools to map and analyze bisulfite treated short reads [17,18,27]. We calculated the correlation between the average methylation of each target, instead of the single corresponding CpG, to the 450k result yielding a median correlation of 0.89 with a median R2 of 0.79
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