Nucleic Acids Research Advance Access published online on November 4, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn691
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Methods Online |
Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies
1Department of Biology, 2Department of Genome Sciences and 3Center on Human Development and Disability, University of Washington, Box 351800, Seattle, WA 98195, USA
*To whom correspondence should be addressed. Tel: +1 206 616 9385; Fax: +1 206 543 3041; Email: genereux{at}u.washington.edu
Received July 31, 2008. Revised September 24, 2008. Accepted September 25, 2008.
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.