Bisulfite-Converted DNA Quantity Evaluation: A Multiplex Quantitative Real-Time PCR System for Evaluation of Bisulfite Conversion

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(R) Bisulfite Conversion System, EpiJET Bisulfite Conversion Kit, EpiTect Fast DNA Bisulfite Kit, and NEBNext(R) 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.

Automated summary

This study introduces a multiplex quantitative real-time PCR system named BisQuE, which can simultaneously analyze three key features of BS conversion, including efficiency, recovery, and degradation levels. Six BS conversion kits were tested, with NEBNext kit showing the lowest conversion efficiency but also the lowest degradation level among them.