Evaluation of whole-genome DNA methylation sequencing library preparation protocols.
Evaluation of whole-genome DNA methylation sequencing library preparation protocols.
复制标题
全基因组DNA甲基化测序文库制备方案的评估。
DOI：
10.1186/s13072-021-00401-y
复制
发表时间：
2021-06-19
影响因子：
3.9
通讯作者：
Shen H
中科院分区：
文献类型：
--
作者：
Morrison J;Koeman JM;Johnson BK;Foy KK;Beddows I;Zhou W;Chesla DW;Rossell LL;Siegwald EJ;Adams M;Shen H
随着测序成本的迅速下降，全基因组DNA甲基化测序的普及率一直在上升。目前存在多种文库制备方案。我们已经在快速冷冻的人类样本上进行了22次全基因组DNA甲基化测序实验，并广泛地对全基因组DNA甲基化测序的常见文库制备方案进行了基准测试，包括三种传统的基于亚硫酸氢盐的方案和一种新的基于酶的方案。此外，比较了两种与减少的起始量相容的试剂盒的不同输入DNA量。此外，我们还提供了用于这些文库类型中的每一种的测序数据的生物信息学分析管道。收集每个试剂盒的各种指标，包括原始读数统计、文库质量和均匀性指标、胞嘧啶保留和技术重复之间的CpG β值一致性。总体而言，NEBNext酶促甲基测序和Swift NGS甲基测序试剂盒在定量上优于其他两种方案。此外，NEB和Swift试剂盒在低输入量下表现良好，验证了它们在DNA是限制因素的应用中的实用性。NEBNext酶促甲基测序试剂盒似乎是高质量DNA全基因组DNA甲基化测序的最佳选择，紧随其后的是Swift试剂盒，它可能对降解样品更有效。此外，一般的生物信息学流水线适用于四种方案，除了Swift Biosciences的NGS-NGS甲基-Seq方案需要额外的修剪以去除Adaptase序列。在线版本包含补充材料，可通过10.1186/s13072-021-00401-y获得。
With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types. An assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor. The NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences’s Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence. The online version contains supplementary material available at 10.1186/s13072-021-00401-y.
登录
查看更多内容
影响因子：
16.6
作者：
Labidi-Galy SI;Papp E;Hallberg D;Niknafs N;Adleff V;Noe M;Bhattacharya R;Novak M;Jones S;Phallen J;Hruban CA;Hirsch MS;Lin DI;Schwartz L;Maire CL;Tille JC;Bowden M;Ayhan A;Wood LD;Scharpf RB;Kurman R;Wang TL;Shih IM;Karchin R;Drapkin R;Velculescu VE
通讯作者：
Velculescu VE
影响因子：
30.8
作者：
Landan, Gilad;Cohen, Netta Mendelson;Tanay, Amos
通讯作者：
Tanay, Amos
影响因子：
3.9
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
通讯作者：
Clark SJ
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
14.8
作者：
Clark, Susan J.;Statham, Aaron;Frommer, Marianne
通讯作者：
Frommer, Marianne