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Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies.

Genereux DP, Johnson WC, Burden AF, Stöger R, Laird CD - Nucleic Acids Res. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Washington, Seattle, WA 98195, USA. genereux@u.washington.edu

ABSTRACT
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.

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Single-stranded oligonucleotides exposed to HighMT bisulfite. Distributions of failed-conversion counts for molecules treated in HighMT bisulfite for 5 min (a), 15 min (b), 30 min (c), 80 min (d) and 200 min (e). We use n to indicate the number of molecules examined; conversion failures are summarized by the mean number of failed-conversion events per molecule, and the SD on this mean. Sunflower plots of individual-molecule failed- and inappropriate-conversion counts for molecules treated with HighMT bisulfite for 5 min (f), 15 min (g), 30 min (h), 80 min (i) and 200 min (j). A single point indicates one molecule, a line indicates two and a z-petaled ‘flower’ indicates z-molecules.
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Figure 4: Single-stranded oligonucleotides exposed to HighMT bisulfite. Distributions of failed-conversion counts for molecules treated in HighMT bisulfite for 5 min (a), 15 min (b), 30 min (c), 80 min (d) and 200 min (e). We use n to indicate the number of molecules examined; conversion failures are summarized by the mean number of failed-conversion events per molecule, and the SD on this mean. Sunflower plots of individual-molecule failed- and inappropriate-conversion counts for molecules treated with HighMT bisulfite for 5 min (f), 15 min (g), 30 min (h), 80 min (i) and 200 min (j). A single point indicates one molecule, a line indicates two and a z-petaled ‘flower’ indicates z-molecules.

Mentions: Our results confirm the finding of Shirashi and Hayatsu (16) that the conversion of unmethylated cytosines in single-stranded DNA occurs very rapidly under HighMT treatment. We found that 92.6% (100−7.4) conversion of unmethylated cytosines was achieved after only 30 min of treatment (Table 3). At 80 and 200 min, conversion of unmethylated cytosines was complete (980 of 980 total unmethylated cytosines; 100%, CI = 99.7–100%) (Figure 4d and e). This indicated that the final 120 min of exposure were unnecessary.Figure 4.


Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies.

Genereux DP, Johnson WC, Burden AF, Stöger R, Laird CD - Nucleic Acids Res. (2008)

Single-stranded oligonucleotides exposed to HighMT bisulfite. Distributions of failed-conversion counts for molecules treated in HighMT bisulfite for 5 min (a), 15 min (b), 30 min (c), 80 min (d) and 200 min (e). We use n to indicate the number of molecules examined; conversion failures are summarized by the mean number of failed-conversion events per molecule, and the SD on this mean. Sunflower plots of individual-molecule failed- and inappropriate-conversion counts for molecules treated with HighMT bisulfite for 5 min (f), 15 min (g), 30 min (h), 80 min (i) and 200 min (j). A single point indicates one molecule, a line indicates two and a z-petaled ‘flower’ indicates z-molecules.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602783&req=5

Figure 4: Single-stranded oligonucleotides exposed to HighMT bisulfite. Distributions of failed-conversion counts for molecules treated in HighMT bisulfite for 5 min (a), 15 min (b), 30 min (c), 80 min (d) and 200 min (e). We use n to indicate the number of molecules examined; conversion failures are summarized by the mean number of failed-conversion events per molecule, and the SD on this mean. Sunflower plots of individual-molecule failed- and inappropriate-conversion counts for molecules treated with HighMT bisulfite for 5 min (f), 15 min (g), 30 min (h), 80 min (i) and 200 min (j). A single point indicates one molecule, a line indicates two and a z-petaled ‘flower’ indicates z-molecules.
Mentions: Our results confirm the finding of Shirashi and Hayatsu (16) that the conversion of unmethylated cytosines in single-stranded DNA occurs very rapidly under HighMT treatment. We found that 92.6% (100−7.4) conversion of unmethylated cytosines was achieved after only 30 min of treatment (Table 3). At 80 and 200 min, conversion of unmethylated cytosines was complete (980 of 980 total unmethylated cytosines; 100%, CI = 99.7–100%) (Figure 4d and e). This indicated that the final 120 min of exposure were unnecessary.Figure 4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Washington, Seattle, WA 98195, USA. genereux@u.washington.edu

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus