Identification and resolution of artifacts in bisulfite sequencing.
Bisulfite sequencing has become the most widely used application to detect 5-methylcytosine (5-MeC) in DNA, and provides a reliable way of detecting any methylated cytosine at single-molecule resolution in any sequence context. The process of bisulfite treatment exploits the different sensitivity of cytosine and 5-MeC to deamination by bisulfite under acidic conditions, in which cytosine undergoes conversion to uracil while 5-MeC remains unreactive. In this article, we address the more commonly encountered experimental artifacts associated with bisulfite sequencing, and provide methods for the detection and elimination of these artifacts. In particular, we focus on conditions that inhibit complete bisulfite-mediated conversion of cytosines in a target sequence, and demonstrate the necessity of complete protein removal from DNA samples prior to bisulfite treatment. We also include a brief summary of the experimental protocol for bisulfite treatment and tips for designing polymerase chain reaction (PCR) primers to amplify from bisulfite-treated DNA.
Warnecke
Peter M
PM
Sydney Cancer Centre, Kanematsu Laboratories, Royal Prince Alfred Hospital, Missenden Road, Camperdown, New South Wales 2050, Australia.
Stirzaker
Clare
C
Song
Jenny
J
Grunau
Christoph
C
Melki
John R
JR
Clark
Susan J
SJ
D044503:5-Methylcytosine; D016477:Artifacts; D003596:Cytosine / Q000031:analogs & derivatives* / Q000032:analysis; D017931:DNA Primers; D005189:False Positive Reactions; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013447:Sulfites / Q000378:metabolism*
D017931:DNA Primers; D013447:Sulfites; D044503:5-Methylcytosine; D003596:Cytosine