Hong Sae Rom, Shin Kyoung-Jin
Department of Forensic Medicine and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.
Front Genet. 2021 Feb 25;12:618955. doi: 10.3389/fgene.2021.618955. eCollection 2021.
Bisulfite (BS) conversion, which includes a series of chemical reactions using bisulfite, is a prerequisite to most DNA methylation analysis methods, and thus is an essential step in the associated research process. Unfortunately, BS conversion leads to the degradation or loss of DNA, which can hinder further downstream analysis. In addition, it is well known that incomplete BS conversion is crucial, as it causes an exaggeration of the DNA methylation level, which can adversely affect the results. Therefore, there have been many attempts to measure three key features of BS conversion: BS conversion efficiency, recovery, and degradation level. In this study, a multiplex quantitative real-time PCR system named BisQuE was suggested to simultaneously analyze three important aspects of the conversion step. By adopting cytosine-free PCR primers for two differently sized multicopy regions, the short amplicon and long amplicon were obtained from both the genomic and BS-converted DNA, thus enabling the obtaining of reliable and sensitive results and the calculation of the degradation level of the conversion step. Also, probes for detecting converted/unconverted templates and C-T indicators for inducing the formula were included in this assay to quantify BS-converted DNA in order to compute the conversion efficiency and recovery. Six BS conversion kits (EZ DNA Methylation-Lightning Kit, Premium Bisulfite kit, MethylEdge Bisulfite Conversion System, EpiJET Bisulfite Conversion Kit, EpiTect Fast DNA Bisulfite Kit, and NEBNext Enzymatic Methyl-seq Conversion Module) were tested in 20 samples using 50 ng of genomic DNA as an input with the BisQuE. The conversion efficiency, degradation levels, as well as recovery rates of the kits were investigated. A total of 99.61-99.90% conversion efficiency was perceived for five of the kits, while the NEBNext kit showed about 94%. The lowest degradation level was shown by the NEBNext kit, whereas the other kits were quite similar. The recovery rates of the kits were found to be within the range of 18-50%. A Qubit assay was also used to compare the recovery rate of BisQuE.
亚硫酸氢盐（BS）转化包括一系列使用亚硫酸氢盐的化学反应，是大多数DNA甲基化分析方法的前提条件，因此是相关研究过程中的关键步骤。不幸的是，BS转化会导致DNA降解或丢失，这可能会阻碍进一步的下游分析。此外，众所周知，不完全的BS转化至关重要，因为它会导致DNA甲基化水平的夸大，从而可能对结果产生不利影响。因此，人们进行了许多尝试来测量BS转化的三个关键特征：BS转化效率、回收率和降解水平。在本研究中，提出了一种名为BisQuE的多重定量实时PCR系统，用于同时分析转化步骤的三个重要方面。通过采用针对两个不同大小的多拷贝区域的无胞嘧啶PCR引物，从基因组DNA和经BS转化的DNA中均获得了短扩增子和长扩增子，从而能够获得可靠且灵敏的结果，并计算转化步骤的降解水平。此外，该检测方法还包括用于检测转化/未转化模板的探针和用于推导公式的C-T指示剂，以对经BS转化的DNA进行定量，从而计算转化效率和回收率。使用BisQuE对六个BS转化试剂盒（EZ DNA甲基化-闪电试剂盒、高级亚硫酸氢盐试剂盒、MethylEdge亚硫酸氢盐转化系统、EpiJET亚硫酸氢盐转化试剂盒、EpiTect快速DNA亚硫酸氢盐试剂盒和NEBNext酶促甲基化测序转化模块）进行了测试，以50 ng基因组DNA作为输入，对20个样本进行检测。研究了各试剂盒的转化效率、降解水平以及回收率。五个试剂盒的转化效率在99.61%-99.90%之间，而NEBNext试剂盒的转化效率约为94%。NEBNext试剂盒的降解水平最低，而其他试剂盒的降解水平相当。各试剂盒的回收率在18%-50%范围内。还使用了Qubit检测法来比较BisQuE的回收率。
