In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×).
Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.
在这项研究中,我们提供优化的WGBS方法,从文库制备到测序和数据处理,为了使HiSeqXTen的每个单通道的全基因组覆盖率达到16-20倍,HCS3.3.76.为了处理和分析数据,我们开发了一个WGBS管道(MET10X),它速度快,可以调用SNP。我们对来自福尔马林固定的石蜡包埋组织的高质量完整DNA和降解DNA进行了WGBS。首先,我们在HiSeq2500平台上比较了不同的文库制备方法,以确定在HiSeqXTen上测序的最佳方法。第二,我们优化了PhiX和基因组尖峰,以在HiSeqXTen上实现更高质量和更高的WGBS数据覆盖率。第三,我们在HiSeqXTen的单道中对同一DNA样本进行了整合全基因组测序(WGS)和WGBS,以提高数据输出.最后,我们比较了HiSeq2500和HiSeqXTen的甲基化数据,发现高度一致性(Pearsonr>0.9×)。
我们一起提供一个系统的,在HiSeqXTen上执行和分析WGBS的有效和完整的方法。我们的协议允许在HiSeqXTen平台上以合理的处理时间和成本进行大规模的WGBS研究。