Identification and resolution of artifacts in bisulfite sequencing
Introduction
The detection of 5-methylcytosine (5-MeC) using bisulfite conversion was first demonstrated by Frommer et al. [1] and Clark et al. [2]. Since that time, the validity of bisulfite sequencing has been confirmed to the point where methods based around bisulfite conversion account for the majority of new data on DNA methylation. Substantial effort has gone into determining the optimal conditions for the “standard” bisulfite reaction as well as specialized modifications for particular templates or source materials (reviewed in [3]). As with any method, bisulfite sequencing has associated technical difficulties and potential artifacts. It should be emphasized that in most circumstances, the bisulfite reaction is quite robust, and the methodology has evolved to a point where the more common problems have been identified and guidelines to avoid or minimize such problems developed. The details of experimental parameters and primer design have been set out in our previous publications [2], [4], [5], [6], and a brief summary of the “standard” protocol appears at the end of this article. The main part of this article focuses on some of the more problematic artifacts associated with interpreting bisulfite sequencing data.
Section snippets
Efficiency of bisulfite conversion
The conversion of cytosine to uracil by bisulfite is remarkably selective and efficient when carried out under standard protocols. In fact, the rate of chemical conversion has been estimated to be on the order of 99.5–99.7% [6], [7] of all cytosines but is more often 95–98%. For most applications this rate of conversion is more than sufficient, and the low level of unconverted cytosines is detectable only by a detailed analysis of cloned fragments. However, if the rate of conversion is less
Origin of false-positive 5-MeC in the bisulfite reaction
There are several independent mechanisms by which false-positive 5-MeC may be apparent after bisulfite treatment, and many of these mechanisms are able to be influenced by alterations to the design of experiments.
Quantitation of 5-MeC in the bisulfite reaction
In addition to artifacts introduced through lack of conversion, the PCR amplification step can also introduce errors that may complicate interpretation of bisulfite sequencing data.
Standard bisulfite treatment protocol
The following protocol has been successfully used in our laboratory to obtain complete bisulfite conversion of a wide range of plasmid and genomic DNAs. For a more extensive analysis of the reaction parameters and PCR primer design, refer to Clark et al. [2] and Grunau et al. [6].
1. Digest the target DNA with a restriction enzyme that does not cut within the sequence of interest, or shear the DNA by passing through a narrow-gauge needle. Typically we use 50 ng to of genomic DNA in a volume
Design of PCR primers to bisulfite-treated DNA
Bisulfite-treated DNA is relatively fragmented and has an unusual base composition, and so does not amplify as readily as untreated DNA. The objective in designing primers for quantitative methylation analysis is to amplify methylated and unmethylated sequences with equal efficiency, while avoiding amplification of any unconverted sequences. Alternately, primers can be designed to amplify only methylated sequences (MS-PCR) [26]; however, care must also be taken using this approach to ensure
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