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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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Conversion errors can distinguish between clonal and unique sequences. (a) Three clonal sequences from end-coded, single-stranded oligonucleotides (Burden et al., manuscript in preparation). All three sequences bear the expected GTATGTGT batchstamp, indicating that they are from the intended experiment. However, they have the same barcode, TTAGATA, indicating that they are clones. Their shared pattern of failed-conversion events confirms their common origin from a single template molecule. (b) Three unique sequences from end-coded, single-stranded oligonucleotides. Each sequence bears the GTATGTGT batchstamp (bold-faced) used in this experiment, and a unique barcode. These sequences also differ in their patterns of failed-conversion events (dark gray boxes), confirming that they proceeded through the conversion and amplification processes independently.
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Figure 13: Conversion errors can distinguish between clonal and unique sequences. (a) Three clonal sequences from end-coded, single-stranded oligonucleotides (Burden et al., manuscript in preparation). All three sequences bear the expected GTATGTGT batchstamp, indicating that they are from the intended experiment. However, they have the same barcode, TTAGATA, indicating that they are clones. Their shared pattern of failed-conversion events confirms their common origin from a single template molecule. (b) Three unique sequences from end-coded, single-stranded oligonucleotides. Each sequence bears the GTATGTGT batchstamp (bold-faced) used in this experiment, and a unique barcode. These sequences also differ in their patterns of failed-conversion events (dark gray boxes), confirming that they proceeded through the conversion and amplification processes independently.

Mentions: We illustrate this point with six sequences collected through PCR amplification of end-coded oligonucleotides. Like mammalian DNA, these oligonucleotides have methyl groups at cytosines within CpG dinucleotides, but not on other cytosines. As we describe in previous sections, these molecules were first encoded with a molecular batchstamp, and a set of random barcodes, and then subjected to bisulfite conversion (Figure 13). The three sequences in Figure 13a all contain the same, intended batch-stamp, indicating that they are not contaminants. However, these three sequences bear an identical barcode, indicating that they are almost certainly copies of a single template molecule. Their identical patterns of failed-conversion events are consistent with the information from their molecular codes, and indicate that these sequences are clones of one another, and should be considered as one molecule—not three—in the analysis of these data.Figure 13.


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)

Conversion errors can distinguish between clonal and unique sequences. (a) Three clonal sequences from end-coded, single-stranded oligonucleotides (Burden et al., manuscript in preparation). All three sequences bear the expected GTATGTGT batchstamp, indicating that they are from the intended experiment. However, they have the same barcode, TTAGATA, indicating that they are clones. Their shared pattern of failed-conversion events confirms their common origin from a single template molecule. (b) Three unique sequences from end-coded, single-stranded oligonucleotides. Each sequence bears the GTATGTGT batchstamp (bold-faced) used in this experiment, and a unique barcode. These sequences also differ in their patterns of failed-conversion events (dark gray boxes), confirming that they proceeded through the conversion and amplification processes independently.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 13: Conversion errors can distinguish between clonal and unique sequences. (a) Three clonal sequences from end-coded, single-stranded oligonucleotides (Burden et al., manuscript in preparation). All three sequences bear the expected GTATGTGT batchstamp, indicating that they are from the intended experiment. However, they have the same barcode, TTAGATA, indicating that they are clones. Their shared pattern of failed-conversion events confirms their common origin from a single template molecule. (b) Three unique sequences from end-coded, single-stranded oligonucleotides. Each sequence bears the GTATGTGT batchstamp (bold-faced) used in this experiment, and a unique barcode. These sequences also differ in their patterns of failed-conversion events (dark gray boxes), confirming that they proceeded through the conversion and amplification processes independently.
Mentions: We illustrate this point with six sequences collected through PCR amplification of end-coded oligonucleotides. Like mammalian DNA, these oligonucleotides have methyl groups at cytosines within CpG dinucleotides, but not on other cytosines. As we describe in previous sections, these molecules were first encoded with a molecular batchstamp, and a set of random barcodes, and then subjected to bisulfite conversion (Figure 13). The three sequences in Figure 13a all contain the same, intended batch-stamp, indicating that they are not contaminants. However, these three sequences bear an identical barcode, indicating that they are almost certainly copies of a single template molecule. Their identical patterns of failed-conversion events are consistent with the information from their molecular codes, and indicate that these sequences are clones of one another, and should be considered as one molecule—not three—in the analysis of these data.Figure 13.

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