Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies.
Bisulfite treatment can be used to ascertain the methylation states of individual cytosines in DNA. Ideally, bisulfite treatment deaminates unmethylated cytosines to uracils, and leaves 5-methylcytosines unchanged. Two types of bisulfite-conversion error occur: inappropriate conversion of 5-methylcytosine to thymine, and failure to convert unmethylated cytosine to uracil. Conventional bisulfite treatment requires hours of exposure to low-molarity, low-temperature bisulfite ('LowMT') and, sometimes, thermal denaturation. An alternate, high-molarity, high-temperature ('HighMT') protocol has been reported to accelerate conversion and to reduce inappropriate conversion. We used molecular encoding to obtain validated, individual-molecule data on failed- and inappropriate-conversion frequencies for LowMT and HighMT treatments of both single-stranded and hairpin-linked oligonucleotides. After accounting for bisulfite-independent error, we found that: (i) inappropriate-conversion events accrue predominantly on molecules exposed to bisulfite after they have attained complete or near-complete conversion; (ii) the HighMT treatment is preferable because it yields greater homogeneity among sites and among molecules in conversion rates, and thus yields more reliable data; (iii) different durations of bisulfite treatment will yield data appropriate to address different experimental questions; and (iv) conversion errors can be used to assess the validity of methylation data collected without the benefit of molecular encoding.
Genereux
Diane P
DP
Department of Biology, University of Washington, Seattle, WA 98195, USA. genereux@u.washington.edu
Johnson
Winslow C
WC
Burden
Alice F
AF
Stöger
Reinhard
R
Laird
Charles D
CD
D044503:5-Methylcytosine / Q000737:chemistry*; D003596:Cytosine / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D004277:DNA, Single-Stranded / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013447:Sulfites / Q000737:chemistry*; D013696:Temperature
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D013447:Sulfites; D044503:5-Methylcytosine; D003596:Cytosine; D004247:DNA; C042345:hydrogen sulfite