Combating PCR Bias in Bisulfite-Based Cytosine Methylation Analysis. Betaine-Modified Cytosine Deamination PCR

Abstract

Sodium bisulfite-induced cytosine deamination/PCR (CD-PCR) is currently the most sensitive and robust method to determine the methylation status of all cytosines in a specific DNA sequence. The CDPCR products are directly sequenced with Thermosequenase and capillary electrophoresis; peak areas are then used to determine the mole fraction of methylated cytosines at each site in a single analysis. Here we show that, if the original DNA sample is a mixture of methylated and unmethylated DNA, conventional CDPCR discriminates against the sequence originating from the methylated DNA; CDPCR product does not accurately represent the methylation status of the original DNA sample. While CDPCR bias can lead to serious errors when determining methylation levels, the addition of betaine (N,N,N-trimethylglycine) to the PCR reaction buffer reduces this bias to less than 10%.

Selected imprinting of INS in the marsupial

“…A 3-minute extension time in the PCR thermal cycling was used to reduce PCR bias by permitting the polymerase to read through CG-rich regions. As no SNP was available to test for experimental biases at the TH-INS TSS, a solution containing 5 mg/mlBSA and 5% glycerol was added to the PCR reactions as a denaturant to reduce PCR bias during amplification [ 88 ]. PCR products were cloned as described above and sequences were analysed using the Quma quantification tool for methylation analysis [ 89 ].…”

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

“…the incomplete cytosine conversion by sodium bisulfite and over-conversion of 5mC, found to be affected by factors like DNA quality, quantity and purification procedures, BS incubation length and temperatures, strand reannealing, polymerase, sequencing errors as well as conversion resistant sequences [2,3,[5][6][7][8]. Different solutions to these biases and artefacts have been proposed, which improved quantitation of DNA methylation at specific loci by PCR and cloning-based methods [2,3,5,7,[9][10][11][12][13][14][15]. Only some of these considerations, however, remain relevant for NGS-based approaches (e.g.…”

Single molecule and single cell epigenomics

“…Additional methods have been developed to assess epigenetically influenced chromatin states diagnostic for transcriptionally regulatory domains including DNAse-seq [34], FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) [35] and Sono-seq [36]. Among the technical challenges associated with these methods are biased amplification of bisulfite treated DNAs [37, 38] and the problem of antibody quality for ChIP studies. Antibody specificity can limit ChIP-seq accuracy and reproducibility.…”