Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten.
Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten.
复制标题
DOI：
10.1186/s13072-018-0194-0
复制
发表时间：
2018-05-28
影响因子：
3.9
通讯作者：
Clark SJ
中科院分区：
文献类型：
--
作者：
Nair SS;Luu PL;Qu W;Maddugoda M;Huschtscha L;Reddel R;Chenevix-Trench G;Toso M;Kench JG;Horvath LG;Hayes VM;Stricker PD;Hughes TP;White DL;Rasko JEJ;Wong JJ;Clark SJ
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）被认为是以单碱基分辨率测定全基因组DNA甲基化的金标准。然而，实现最佳覆盖深度的高测序成本限制了其在基础和临床研究中的应用。为了实现15×人类甲基化组的覆盖，使用WGBS，需要大约三个100 bp配对末端Illumina HiSeq 2500测序泳道。因此，重要的是要开发测序技术的进步，以实现具有成本效益的高覆盖率测序。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，使HiSeq X Ten，HCS 3.3.76的每个单泳道能够实现16-20×全基因组覆盖。为了处理和分析数据，我们开发了一个快速且可以调用SNP的WGBS管道（METH 10 X）。我们对福尔马林固定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HiSeq 2500平台上比较了不同的文库制备方法，以确定在HiSeq X Ten上测序的最佳方法。其次，我们优化了PhiX和基因组加标，以实现HiSeq X Ten上WGBS数据的更高质量和覆盖率。第三，我们在HiSeq X Ten的单个泳道中对同一DNA样品进行了整合的全基因组测序（WGS）和WGBS，以提高数据输出。最后，我们比较了HiSeq 2500和HiSeq X Ten的甲基化数据，发现高度一致性（Pearson r > 0.9×）。我们共同提供了一种系统、高效和完整的方法来在HiSeq X Ten上执行和分析WGBS。我们的方案允许在HiSeq X Ten平台上以合理的处理时间和成本进行大规模WGBS研究。本文的在线版本（10.1186/s13072-018-0194-0）包含补充材料，可供授权用户使用。
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. 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. The online version of this article (10.1186/s13072-018-0194-0) contains supplementary material, which is available to authorized users.
登录
查看更多内容
影响因子：
4.4
作者：
Bibikova, Marina;Barnes, Bret;Shen, Richard
通讯作者：
Shen, Richard
影响因子：
30.8
作者：
Kulis M;Merkel A;Heath S;Queirós AC;Schuyler RP;Castellano G;Beekman R;Raineri E;Esteve A;Clot G;Verdaguer-Dot N;Duran-Ferrer M;Russiñol N;Vilarrasa-Blasi R;Ecker S;Pancaldi V;Rico D;Agueda L;Blanc J;Richardson D;Clarke L;Datta A;Pascual M;Agirre X;Prosper F;Alignani D;Paiva B;Caron G;Fest T;Muench MO;Fomin ME;Lee ST;Wiemels JL;Valencia A;Gut M;Flicek P;Stunnenberg HG;Siebert R;Küppers R;Gut IG;Campo E;Martín-Subero JI
通讯作者：
Martín-Subero JI
影响因子：
46.9
作者：
通讯作者：
--
无细胞DNA及其细胞促进器的整个基因组硫酸氢盐测序可发现胎盘降低甲基化结构域。
影响因子：
12.3
作者：
Jensen TJ;Kim SK;Zhu Z;Chin C;Gebhard C;Lu T;Deciu C;van den Boom D;Ehrich M
通讯作者：
Ehrich M
影响因子：
64.8
作者：
通讯作者：
--