Pabinger Stephan, Ernst Karina, Pulverer Walter, Kallmeyer Rainer, Valdes Ana M, Metrustry Sarah, Katic Denis, Nuzzo Angelo, Kriegner Albert, Vierlinger Klemens, Weinhaeusel Andreas
Health & Environment Department, Molecular Diagnostics, AIT-Austrian Institute of Technology, Vienna, Austria.
The Department of Twin Research & Genetic Epidemiology, King's College London, St Thomas' Campus, London, United Kingdom.
PLoS One. 2016 Jul 28;11(7):e0160227. doi: 10.1371/journal.pone.0160227. eCollection 2016.
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/.
对亚硫酸氢盐脱氨处理后的DNA所生成的PCR扩增子进行靶向测序，是一种灵活且经济高效的方法，可在单CpG分辨率下研究样本的甲基化情况，并进行后续的多靶点、多样本比较。目前，尚无针对个人基因组测序仪（PGM）进行靶向亚硫酸氢盐测序的特定平台协议、支持或分析解决方案。在此，我们展示了一种名为TABSAT的新型工具，用于分析在Ion Torrent测序仪上生成的靶向亚硫酸氢盐测序数据。该工作流程从原始测序数据开始，进行质量评估，并使用定制版的Bismark将 reads 映射到参考基因组。该流程将结果可视化为棒棒糖图，并能够推断样本中存在的特定甲基化模式。然后对获得的图谱进行汇总并在样本之间进行比较。为了评估靶向亚硫酸氢盐测序工作流程的性能，使用了48个样本，从每个样本中生成53种不同的亚硫酸氢盐测序PCR扩增子，从而得到2544个扩增子靶点。使用1196822条比对 reads，我们获得了平均282X的覆盖度。接下来，我们将这些靶点的测序结果与Illumina 450k甲基化芯片上相应位点的甲基化水平进行了比较。计算得出的平均皮尔逊相关系数为0.91，这与行业领先的CpG甲基化平台之一的测序结果相符，表明靶向扩增子亚硫酸氢盐测序为DNA甲基化研究提供了一种准确且经济高效的方法，例如可用于对Illumina Infinium 450k甲基化数据进行独立于平台的验证。TABSAT提供了一种分析Ion Torrent仪器生成数据的新方法，也可用于Illumina MiSeq平台的数据。它可以通过Platomics平台轻松访问，该平台提供基于网络的图形用户界面以及样本和参数存储功能。TABSAT在GNU通用公共许可证第3.0版（GPLv3）下可从https://github.com/tadkeys/tabsat/和http://demo.platomics.com/免费获取。
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