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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 DNA treated under HighMT conditions. Cytosine site-specific failed-conversion counts for HighMT-treated, single-stranded DNA examined after 5 min (a), 15 min (b) and 30 min (c). Fraction of unmethylated cytosines surviving at two relatively rapidly converted sites (1 and 2), and two relatively slowly converted sites (7 and 9) after treatment for 5–200 min (d). We use n to indicate the total number of sequences examined.
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Figure 9: Single-stranded DNA treated under HighMT conditions. Cytosine site-specific failed-conversion counts for HighMT-treated, single-stranded DNA examined after 5 min (a), 15 min (b) and 30 min (c). Fraction of unmethylated cytosines surviving at two relatively rapidly converted sites (1 and 2), and two relatively slowly converted sites (7 and 9) after treatment for 5–200 min (d). We use n to indicate the total number of sequences examined.

Mentions: The HighMT treatment, as compared to the LowMT treatment, yielded remarkably little variation among sites in the time to complete conversion (Figure 9). Although sites differed somewhat in their frequencies of conversion when examined early in the treatment, by 30 min, sampled molecules contained no evidence of site-site variation in failed-conversion counts (P = 0.15). The time course of conversion observed was similar for sites 1 and 2, which were converted fairly rapidly, and sites 7 and 9, which were comparatively slow in their conversion (Figure 9d).Figure 9.


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 DNA treated under HighMT conditions. Cytosine site-specific failed-conversion counts for HighMT-treated, single-stranded DNA examined after 5 min (a), 15 min (b) and 30 min (c). Fraction of unmethylated cytosines surviving at two relatively rapidly converted sites (1 and 2), and two relatively slowly converted sites (7 and 9) after treatment for 5–200 min (d). We use n to indicate the total number of sequences examined.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 9: Single-stranded DNA treated under HighMT conditions. Cytosine site-specific failed-conversion counts for HighMT-treated, single-stranded DNA examined after 5 min (a), 15 min (b) and 30 min (c). Fraction of unmethylated cytosines surviving at two relatively rapidly converted sites (1 and 2), and two relatively slowly converted sites (7 and 9) after treatment for 5–200 min (d). We use n to indicate the total number of sequences examined.
Mentions: The HighMT treatment, as compared to the LowMT treatment, yielded remarkably little variation among sites in the time to complete conversion (Figure 9). Although sites differed somewhat in their frequencies of conversion when examined early in the treatment, by 30 min, sampled molecules contained no evidence of site-site variation in failed-conversion counts (P = 0.15). The time course of conversion observed was similar for sites 1 and 2, which were converted fairly rapidly, and sites 7 and 9, which were comparatively slow in their conversion (Figure 9d).Figure 9.

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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Related in: MedlinePlus