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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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An end-coded TM oligonucleotide ligated while base-paired with BU. The 5-methylcytosines are present at 10 CpG sites on the top strand, TM, and are indicated with ‘Me’. The end-coder (Burden et al., manuscript in preparation) contains a batchstamp common to all molecules processed in a given experiment, and a randomly generated barcode. The end-coder is attached to the top, TM, but not the bottom, BU, strand of TM:BU. Thus, the bottom strand will separate from the top strand under denaturating conditions. Oligonucleotides labeled with end-coder have a forward primer binding site on TM, and a reverse-primer binding site of 21 nt, indicated here in purple, on the end-coder itself (after Figure 1 of Burden et al., manuscript in preparation).
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Figure 3: An end-coded TM oligonucleotide ligated while base-paired with BU. The 5-methylcytosines are present at 10 CpG sites on the top strand, TM, and are indicated with ‘Me’. The end-coder (Burden et al., manuscript in preparation) contains a batchstamp common to all molecules processed in a given experiment, and a randomly generated barcode. The end-coder is attached to the top, TM, but not the bottom, BU, strand of TM:BU. Thus, the bottom strand will separate from the top strand under denaturating conditions. Oligonucleotides labeled with end-coder have a forward primer binding site on TM, and a reverse-primer binding site of 21 nt, indicated here in purple, on the end-coder itself (after Figure 1 of Burden et al., manuscript in preparation).

Mentions: Oligonucleotide molecules to be used in experiments with single-stranded DNA were batchstamped and barcoded through covalent attachment of an ‘end-code’ (Burden et al., manuscript in preparation; Figure 3). Each end-code oligonucleotide contained a defined batchstamp specific to that experiment, a randomly generated barcode, and a 5-nt overhang complementary to the 5′-overhang of the top strand of the double-stranded oligonucleotide. End-coders were combined with annealed TM:BU oligonucleotides at room temperature in a 1:16.7 molar ratio, and treated with T4 ligase, as per the manufacturer's; instructions (NEB, Ipswich, MA). After 1 hour, the ligase was heat-inactivated at 65°C for 20 min. The ligation step is expected to bind the end-coder to the top strand of the annealed oligonucleotide, using the 5′-phosphate of TM. End-coders differ from hairpin linkers in two ways: (i) they bear a reverse-primer binding site and (ii) they lack a 5′-phosphate, thus ensuring that they attach covalently only to the top strand of TM:BU. The end-coded top strand contains both primer binding sites, and thus can be used to detect top-strand conversion errors in the protocol we describe here.Figure 3.


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)

An end-coded TM oligonucleotide ligated while base-paired with BU. The 5-methylcytosines are present at 10 CpG sites on the top strand, TM, and are indicated with ‘Me’. The end-coder (Burden et al., manuscript in preparation) contains a batchstamp common to all molecules processed in a given experiment, and a randomly generated barcode. The end-coder is attached to the top, TM, but not the bottom, BU, strand of TM:BU. Thus, the bottom strand will separate from the top strand under denaturating conditions. Oligonucleotides labeled with end-coder have a forward primer binding site on TM, and a reverse-primer binding site of 21 nt, indicated here in purple, on the end-coder itself (after Figure 1 of Burden et al., manuscript in preparation).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: An end-coded TM oligonucleotide ligated while base-paired with BU. The 5-methylcytosines are present at 10 CpG sites on the top strand, TM, and are indicated with ‘Me’. The end-coder (Burden et al., manuscript in preparation) contains a batchstamp common to all molecules processed in a given experiment, and a randomly generated barcode. The end-coder is attached to the top, TM, but not the bottom, BU, strand of TM:BU. Thus, the bottom strand will separate from the top strand under denaturating conditions. Oligonucleotides labeled with end-coder have a forward primer binding site on TM, and a reverse-primer binding site of 21 nt, indicated here in purple, on the end-coder itself (after Figure 1 of Burden et al., manuscript in preparation).
Mentions: Oligonucleotide molecules to be used in experiments with single-stranded DNA were batchstamped and barcoded through covalent attachment of an ‘end-code’ (Burden et al., manuscript in preparation; Figure 3). Each end-code oligonucleotide contained a defined batchstamp specific to that experiment, a randomly generated barcode, and a 5-nt overhang complementary to the 5′-overhang of the top strand of the double-stranded oligonucleotide. End-coders were combined with annealed TM:BU oligonucleotides at room temperature in a 1:16.7 molar ratio, and treated with T4 ligase, as per the manufacturer's; instructions (NEB, Ipswich, MA). After 1 hour, the ligase was heat-inactivated at 65°C for 20 min. The ligation step is expected to bind the end-coder to the top strand of the annealed oligonucleotide, using the 5′-phosphate of TM. End-coders differ from hairpin linkers in two ways: (i) they bear a reverse-primer binding site and (ii) they lack a 5′-phosphate, thus ensuring that they attach covalently only to the top strand of TM:BU. The end-coded top strand contains both primer binding sites, and thus can be used to detect top-strand conversion errors in the protocol we describe here.Figure 3.

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