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BACKGROUND

Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing.

RESULTS

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×).

CONCLUSIONS

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.

背景

全面的全基因组 DNA 甲基化分析对于深入了解发育和疾病过程中的表观遗传重编程至关重要。在不同的全基因组 DNA 甲基化技术中，全基因组亚硫酸氢盐测序（WGBS）被认为是在单碱基分辨率下检测全基因组 DNA 甲基化的金标准。然而，为了实现最佳覆盖深度，高通量测序的高成本限制了其在基础和临床研究中的应用。为了实现人类甲基组 15×的覆盖，使用 WGBS 需要大约三条 100 碱基对的 Illumina HiSeq 2500 测序通道。因此，开发能够实现经济高效的高通量测序的测序技术的进步非常重要。

结果

在这项研究中，我们提供了一种优化的 WGBS 方法，从文库制备到测序和数据处理，以实现每条 HiSeq X Ten、HCS 3.3.76 通道的 16-20×全基因组覆盖。为了处理和分析数据，我们开发了一种快速的 WGBS 管道（METH10X），能够调用 SNPs。我们对高质量完整的 DNA 以及福尔马林固定石蜡包埋组织中的降解 DNA 进行了 WGBS。首先，我们在 HiSeq 2500 平台上比较了不同的文库制备方法，以确定最适合在 HiSeq X Ten 上测序的方法。其次，我们优化了 PhiX 和基因组 Spike-ins，以提高 HiSeq X Ten 上 WGBS 数据的质量和覆盖度。第三，我们在 HiSeq X Ten 的一条通道中对同一个 DNA 样本进行了全基因组测序（WGS）和 WGBS 的整合，以提高数据输出。最后，我们比较了 HiSeq 2500 和 HiSeq X Ten 上的甲基化数据，发现高度一致性（Pearson r＞0.9×）。