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

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.

Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR.

PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular 'batch-stamps' that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes.

Statistical inference of in vivo properties of human DNA methyltransferases from double-stranded methylation patterns.

DNA methyltransferases establish methylation patterns in cells and transmit these patterns over cell generations, thereby influencing each cell's epigenetic states. Three primary DNA methyltransferases have been identified in mammals: DNMT1, DNMT3A and DNMT3B. Extensive in vitro studies have investigated key properties of these enzymes, namely their substrate specificity and processivity. Here we study these properties in vivo, by applying novel statistical analysis methods to double-stranded DNA methylation patterns collected using hairpin-bisulfite PCR. Our analysis fits a novel Hidden Markov Model (HMM) to the observed data, allowing for potential bisulfite conversion errors, and yields statistical estimates of parameters that quantify enzyme processivity and substrate specificity. We apply this model to methylation patterns established in vivo at three loci in humans: two densely methylated inactive X (Xi)-linked loci (FMR1 and G6PD), and an autosomal locus (LEP), where methylation densities are tissue-specific but moderate. We find strong evidence for a high level of processivity of DNMT1 at FMR1 and G6PD, with the mean association tract length being a few hundred base pairs. Regardless of tissue types, methylation patterns at LEP are dominated by DNMT1 maintenance events, similar to the two Xi-linked loci, but are insufficiently informative regarding processivity to draw any conclusions about processivity at that locus. At all three loci we find that DNMT1 shows a strong preference for adding methyl groups to hemi-methylated CpG sites over unmethylated sites. The data at all three loci also suggest low (possibly 0) association of the de novo methyltransferases, the DNMT3s, and are consequently uninformative about processivity or preference of these enzymes. We also extend our HMM to reanalyze published data on mouse DNMT1 activities in vitro. The results suggest shorter association tracts (and hence weaker processivity), and much longer non-association tracts than human DNMT1 in vivo.

Hemimethylation and non-CpG methylation levels in a promoter region of human LINE-1 (L1) repeated elements.

DNA methylation within the promoter region of human LINE1 (L1) transposable elements is important for maintaining transcriptional inactivation and for inhibiting L1 transposition. Determining methylation patterns on the complementary strands of repeated sequences is difficult using standard bisulfite methylation analysis. Evolutionary changes in each repeat and the variations between cells or alleles of the same repeat lead to a heterogeneous population of sequences. Potential sequence biases can arise during analyses that are different for the converted sense and antisense strands. These problems can be avoided with hairpin-bisulfite PCR, a double-stranded PCR method in which complementary strands of individual molecules are attached by a hairpin linker ligated to genomic DNA. Using human L1 elements to study methylation of repeated sequences, (i) we distinguish valid L1 sequences from redundant and contaminant sequences by applying the powerful new method of molecular barcodes, (ii) we resolve a controversy on the level of hemimethylation of L1 sequences in fetal fibroblasts in favor of relatively little hemimethylation, (iii) we report that human L1 sequences in different cell types also have primarily concordant CpG methylation patterns on complementary strands, and (iv) we provide evidence that non-CpG cytosines within the regions analyzed are rarely methylated.