A MBD-seq protocol for large-scale methylome-wide studies with (very) low amounts of DNA
A MBD-seq protocol for large-scale methylome-wide studies with (very) low amounts of DNA
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DOI：
10.1080/15592294.2017.1335849
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发表时间：
2017-01-01
期刊：
影响因子：
3.7
通讯作者：
van den Oord, Edwin J. C. G.
中科院分区：
文献类型：
--
作者：
Aberg, Karolina A.;Chan, Robin F.;van den Oord, Edwin J. C. G.
我们最近表明，经过优化后，我们的甲基-CpG结合结构域测序(MBD-SEQ)应用程序接近全基因组亚硫酸盐测序(WGB-SEQ)获得的甲基组覆盖范围，但成本足以支持大规模关联研究。MBD-SEQ先前的一个缺点是需要相对大量的基因组DNA(理想情况下为1微克)来获得高质量的数据。生物材料通常收集成本很高，提供的DNA数量有限，而且可能根本不会产生足够的起始材料。使用少量DNA的能力将增加可以进行的研究的广度和数量。因此，我们进一步优化了浓缩步骤。在这种低起始材料方案下，MBD-SEQ的表现与需要充足起始材料(>；1 mg)的方案一样好，甚至更好。仅使用15 ng DNA作为输入，数据质量损失最小，以类似的错误/阳性率达到WGB-SEQ覆盖率的93%(输入DNA的标准量)。此外，在大量基因组特征中，MBD-SEQ甲基化图谱与WGB-SEQ的甲基化图谱密切跟踪，其影响大小甚至略大。这表明MBD-seq提供了关于甲基组的类似信息，并在某种程度上更准确地对甲基化状态进行了分类。当15 ng DNA作为起始材料时，性能会下降，但即使只有5 ng，MBD-SEQ仍然可以达到WGB-SEQ的90%的覆盖率，具有类似的全基因组甲基化特征。因此，建议的方案对于使用(非常)少量DNA进行足够的动力和成本效益的甲基组范围的研究来说是一个有吸引力的选择。
We recently showed that, after optimization, our methyl-CpG binding domain sequencing (MBD-seq) application approximates the methylome-wide coverage obtained with whole-genome bisulfite sequencing (WGB-seq), but at a cost that enables adequately powered large-scale association studies. A prior drawback of MBD-seq is the relatively large amount of genomic DNA (ideally > 1 mu g) required to obtain high-quality data. Biomaterials are typically expensive to collect, provide a finite amount of DNA, and may simply not yield sufficient starting material. The ability to use low amounts of DNA will increase the breadth and number of studies that can be conducted. Therefore, we further optimized the enrichment step. With this low starting material protocol, MBD-seq performed equally well, or better, than the protocol requiring ample starting material (> 1 mg). Using only 15 ng of DNA as input, there is minimal loss in data quality, achieving 93% of the coverage of WGB-seq (with standard amounts of input DNA) at similar false/positive rates. Furthermore, across a large number of genomic features, the MBD-seq methylation profiles closely tracked those observed for WGB-seq with even slightly larger effect sizes. This suggests that MBD-seq provides similar information about the methylome and classifies methylation status somewhat more accurately. Performance decreases with < 15 ng DNA as starting material but, even with as little as 5 ng, MBD-seq still achieves 90% of the coverage of WGB-seq with comparable genome-wide methylation profiles. Thus, the proposed protocol is an attractive option for adequately powered and cost-effective methylome-wide investigations using (very) low amounts of DNA.