Laboratory for Molecular Neuroscience, Department of Psychiatry, Social Psychiatry and Psychotherapy, Hannover Medical School, Hannover, Germany.
Department of Human Genetics, Hannover Medical School, Hannover, Germany.
BMC Med Genomics. 2023 Oct 23;16(1):257. doi: 10.1186/s12920-023-01694-6.
Bisulfite sequencing has long been considered the gold standard for measuring DNA methylation at single CpG resolution. However, in recent years several new approaches like nanopore sequencing have been developed due to hints for a partial error-proneness of bisulfite sequencing. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter from our previous studies obtained by bisulfite sequencing.
We compared methylation rates determined by direct bisulfite sequencing and nanopore sequencing following Cas9-mediated PCR-free enrichment.
We could show that CpG methylation levels above 20% corroborate well with our previous data. Within the range between 0 and 20% methylation, however, Sanger sequencing data have to be interpreted cautiously, at least in the investigated region of interest (TRPA1 promotor region).
Based on the investigation of the TRPA1- region as an example, the present work can help in choosing the right method out of the two current main approaches for methylation analysis for different individual settings regarding many factors like cohort size, costs and prerequisites that should be fulfilled for each method. All in all, both methods have their raison d'être. Furthermore, the present paper contains and illustrates some important basic information and explanation of how guide RNAs should be located for an optimal outcome in Cas9 mediated PCR free target enrichment.
亚硫酸氢盐测序长期以来一直被认为是测量单个 CpG 分辨率下 DNA 甲基化的金标准。然而，近年来，由于提示亚硫酸氢盐测序存在部分易错性，几种新的方法，如纳米孔测序，已经得到了发展。由于这些错误是序列特异性的，我们旨在验证我们之前使用亚硫酸氢盐测序获得的 TRPA1 启动子特定区域的甲基化数据。
我们比较了 Cas9 介导的无 PCR 富集后直接亚硫酸氢盐测序和纳米孔测序确定的甲基化率。
我们可以证明，超过 20%的 CpG 甲基化水平与我们之前的数据很好地吻合。然而，在 0 到 20%的甲基化范围内，Sanger 测序数据至少在研究的感兴趣区域（TRPA1 启动子区域）需要谨慎解释。
基于对 TRPA1 区域的研究，作为一个例子，本工作可以帮助在不同的个体设置下，根据许多因素，如队列大小、成本和每种方法应满足的前提条件，从两种当前主要的甲基化分析方法中选择正确的方法。总的来说，这两种方法都有其存在的理由。此外，本文还包含并说明了一些重要的基本信息和解释，说明如何为 Cas9 介导的无 PCR 目标富集中获得最佳结果定位向导 RNA。
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