Digital bisulfite genomic DNA sequencing overview. An amount of bisulfite-converted DNA was loaded such that individual template DNA molecules were amplified on a 96-well PCR plate. We targeted a maximum of 20−30 molecules to be sequenced in each PCR plate in order to minimize the occurrence of two or more PCR template molecules per reaction well. Positive amplifications are evaluated by SYBR green melting curve analyses, and PCR products from these wells are removed and purified with Exonuclease I and Shrimp Alkaline Phosphatase (Exo-SAP-IT) to remove unused PCR primers and dNTPs. PCR products representing individual DNA molecules are then subject to DNA sequencing.

Digital MethyLight-based real-time PCR amplification. (A) Principle of amplifying individual methylated DNA molecules using digital MethyLight. A bisulfite-converted DNA sample is diluted and divided into multiple PCR reaction wells such that the target methylated DNA molecules are less than one molecule per reaction well. (B) Digital MethyLight was applied to serial dilutions of in vitro methylated DNA. The PITX2 MethyLight reaction for each serial dilution was spread over 96 PCR reaction wells, the fluorescence signals for each dilution were plotted against the PCR cycle number and the number of positives were counted. The approximate number of haploid genomes was also evaluated for each dilution. The dashed vertical line represents the mean cycle threshold (Ct) value of single methylated PITX2 DNA molecules. (C) Comparison of digital and classic MethyLight assay sensitivities. Two identical mixtures of 25 pg M.SssI-DNA plus 50 ng of unmethylated WGA-DNA were each analyzed for PITX2 methylation with one mixture analyzed in one well (classic) and the second analyzed over the remaining 95 wells of a PCR plate (digital). This experiment was analyzed 20 times for each assay. The positive methylated PITX2 molecules are indicated by the black wells and the + symbol indicates a positive signal for each assay. The percentage of assays positive for PITX2 methylation is plotted for both classic and digital MethyLight assays.

Digital MethyLight on plasma samples from 44 breast cancer patients and 13 apparently healthy controls. DNA from 500 µl plasma was purified, bisulfite converted and a DNA amount from 100 µl plasma was subject to digital MethyLight. Each sample was analyzed individually for FOXE1, CLDN5 or RUNX3 methylation, as well as with all three reactions multiplexed. The results are presented as the number of methylated molecules per 100 µl plasma for each sample. The DNA concentration in each plasma sample was estimated based on a TaqMan reaction specific for ALU repetitive elements.

Comparison between conventional and digital bisulfite genomic DNA sequencing of MLH1 CpG island. Bisulfite-converted tumor DNA from two colorectal cancer patients was amplified at the MLH1 locus and subject to (A) TOPO-TA cloning followed by DNA sequencing, and (B) Digital bisulfite genomic DNA sequencing. Each horizontal line represents an individual DNA molecule, and the circles represent CpG dinucleotides. Filled in circles are methylated CpGs while open circles are unmethylated CpGs. The asterisk (*) represents DNA sequencing reads that gave signals for both methylation and the absence of methylation for a specific CpG dinucleotide, and may be indicative of two DNA molecules amplified in the PCR reaction well.

Detection of single methylated PITX2 molecules serial dilutions of bisulfite-converted M.SssI-DNA using microfluidic digital MethyLight (Fluidigm). Each DNA sample was compartmentalized into 1104 reaction chambers of 10 nl each and amplifications were visualized by fluorescence emission in each positive chamber.