Multiplexed DNA Methylation Analysis of Target Regions Using Microfluidics (Fluidigm)

: Whole genome shotgun bisulfite sequencing is a method used to generate genome-wide methylation profiles. There are many available protocols to validate the results of this genome-wide method, but they mostly share the limitation of measuring methylation at a small number of CpG positions in small numbers of samples. We developed a multiplexed DNA methylation analysis protocol, which allows for the simultaneous quantitative measurement of cytosine methylation at single nucleotide resolution in 48 PCR amplicons and 48 samples utilizing the microfluidic system established by Fluidigm. Following bisulfite conversion of 500 ng of the target DNA, a PCR reaction is performed using a 48.48 Access Array, which allows parallel amplification of 48 samples by 48 primer pairs. The products of each reaction are labeled with individual, sample specific tags, pooled in a single library and sequenced using the Illumina MiSeq sequencer. The advantages of this system are: speed, small amount of input material, single nucleotide resolution, high coverage of each locus, low cost of simultaneously assaying multiple CpG loci in multiple DNA samples and high reproducibility.