The bisulfite genomic sequencing protocol

Abstract

The bisulfite genomic sequencing (BGS) proto-col has gained worldwide popularity as the me-thod of choice for analyzing DNA methylation. It is this popular because it is a powerful protocol and it may be coupled with many other applica-tions. However, users often run into a slew of problems, including incomplete conversion, over-ly degraded DNA, sub-optimal PCR amplifica-tions, 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 pro-tocol has worked successfully (>99.9 % conver-sion) 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 PCR reactions. The aim of this article is to make learn-ing and usage of BGS easier, more efficient and standardized for all users