The bisulfite genomic sequencing protocol

Abstract

The bisulfite genomic sequencing (BGS) protocol has gained worldwide popularity as the method of choice for analyzing DNA methylation. It is this popular because it is a powerful protocol and it may be coupled with many other applications. However, users often run into a slew ofproblems, including incomplete conversion,overly degraded DNA, sub-optimal PCR amplifications, false positives, uninformative results, or altogether failed experiments. We pinpoint the reasons why these problems arise and carefully explain the critical steps toward accomplishing a successful experiment step-by-step. This protocol has worked successfully (>99.9% conversion) on as little as 100 ng of DNA derived from nearly 10-year-old DNA samples extracted from whole blood stored at -80°C and resulted in enough converted DNA for more than 50 PCRreactions. The aim of this article is to makelearning and usage of BGS easier, more efficient and standardized for all users.