甲基化捕获测序能否替代450K

任何一个新技术,在蓬勃发展的过程中肯定是少不了大量的测评研究,从技术细节,平台, 软件工具算法多个维度的对比探索。

发表于 Epigenetics . 2016; 链接 https://pubmed.ncbi.nlm.nih.gov/26786415/,文章标题是:《Comparison of Methyl-capture Sequencing vs. Infinium 450K methylation array for methylome analysis in clinical samples》,随着 甲基化技术的深入人心, epigenome-wide association studies (EWAS) 时代到来了,但是临床上的EWAS研究要求成本可控,而且样品DNA起始量低,但是红极一时的450K芯片不尽人意,所以研究者们想试试看Methyl-Capture Sequencing (MC Seq) 能否替代450K。

首先遍历一下其它相似技术的缺点

甲基化技术里面,whole-genome bisulfite conversion (WGBS) 是金标准,但是价格昂贵,数据处理消耗计算资源,而Reduced- representation bisulfite sequencing (RRBS) 和Methylated DNA Immunoprecipitation Sequencing (MeDIP-Seq),都是片面的,只有 MC Seq 克服了它们其它这些技术的缺点:

  • lower genome coverage (Infinium 450K),
  • high cost and processing time (WGBS),
  • overrepre- sentation of repeated (RRBS)
  • methylated regions (MeDIP- Seq).

设计实验来评估

主要是采用 SureSelect Human Methyl-Seq panel (Agilent Technologies) 这个平台做 Methyl-Capture Sequencing (MC Seq) ,纳入7个样品 :

image-20211114153341574

技术指标是都超级好!

首先它自己内部的重复性很好,其次它不同DNA起始量差异也不大。最重要的是它跟最流行的450K基本上也没有差异,如下所示:

image-20211114153438123

可以从以下5个点来比较 (MC Seq vs. Infinium 450K) 是否值得被选用做EWAS研究 :

  • (i) desired genomic coverage (MC Seq has higher density of CpGs, and hence higher coverage of variable sites);
  • (ii) expected inter-group differences in methylation or sensitivity desired (Infinium 450K has higher sensitivity for subtle variations in methylome);
  • (iii) cost (Infinium 450K has lower cost)
  • (iv) coverage of methylation sites in non-CpG context (MC Seq has a higher capacity).
  • The fifth factor for consideration is whether an investigator wishes to customize and specify methylation sites of interest, which can be only done through bait design in MC Seq.

有意思的是作者并没有公布数据,这样的结论很难让人信服啊!难道不能是两个公司打架:

  • Illumina Infinium HumanMethylation450 BeadChips (Illumina Inc., CA, USA)
  • SureSelect Human Methyl-Seq panel (Agilent Technologies)

甲基化技术多如牛毛,我们目前仅仅是提供甲基化芯片数据处理系统性教程。

甲基化背景知识

甲基化测序的 WGBS和RRBS,还有 芯片是最高频的甲基化技术,其中甲基化芯片数据处理我是有视频课程的,首先需要阅读我在生信技能树的甲基化系列教程,目录如下

然后就可以看我在B站免费分享的视频课程《甲基化芯片(450K或者850K)数据处理 》

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