Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region.
Comparison of methylation estimates obtained via MinION nanopore sequencing and sanger bisulfite sequencing in the TRPA1 promoter region.
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DOI：
10.1186/s12920-023-01694-6
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发表时间：
2023-10-23
影响因子：
2.7
通讯作者：
中科院分区：
文献类型：
--
作者：
亚硫酸氢盐测序长期以来一直被认为是在单 CpG 分辨率下测量 DNA 甲基化的金标准。然而，近年来，由于亚硫酸氢盐测序存在部分错误倾向，因此开发了纳米孔测序等几种新方法。由于这些错误被证明是序列特异性的，我们的目的是验证我们之前通过亚硫酸氢盐测序获得的研究中 TRPA1 启动子特定区域的甲基化数据。我们比较了直接亚硫酸氢盐测序和 Cas9 介导的无 PCR 富集后的纳米孔测序测定的甲基化率。我们可以证明 CpG 甲基化水平高于 20%，这与我们之前的数据很好地吻合。然而，在 0 至 20% 甲基化范围内，必须谨慎解释 Sanger 测序数据，至少在所研究的感兴趣区域（TRPA1 启动子区域）是如此。以 TRPA1- 区域的研究为例，目前的工作可以帮助从当前两种主要的甲基化分析方法中选择正确的方法，针对不同的个体环境，考虑到队列大小、成本和每种方法应满足的先决条件等许多因素。总而言之，这两种方法都有其存在的理由。此外，本文包含并说明了一些重要的基本信息和解释，说明如何定位引导 RNA，以在 Cas9 介导的无 PCR 靶标富集中获得最佳结果。在线版本包含可在 10.1186/s12920-023-01694-6 获取的补充材料。
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. The online version contains supplementary material available at 10.1186/s12920-023-01694-6.
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