(740a) Ultrasensitive Microfluidic Assay for Genome-Wide DNA Methylation Analysis and Precision Medicine
To improve the sensitivity of the assay for studying subsets of neurons, we developed a diffusion-based microfluidic assay that preserved significantly more DNA than conventional assays. There were two main factors contributing to the DNA preservation: 1) Taking advantage of manipulating solution with extremely small volume (<1 µl), bisulfite treatment (e.g. dosing, temperature and time) were well controlled. 2) Column-based DNA desalting in conventional assay was replaced by diffusion purification 3. The diffusion scheme also increased the concentration of bisulfite mix which shortened the reaction time from 4~16 hours to 1-2 hours.
We used our microfluidic protocol to perform reduced representative bisulfite sequencing (RRBS) with various amounts of genomic DNA extracted from GM12878 cells. For all 4 sample sizes, replicate experiments were highly correlated (r = 0.995, 0.992, 0.979 and 0.912 for 300, 10, 1 and 0.3 ng, respectively). With similar sequencing depth, we identified averagely 1.82 million CpGs with 5x coverage using 0.3ng DNA, compared to 0.94 million in ENCODE data (deposited standard data using 1-2 µg DNA). When 300 ng samples were used as a reference, 92.1, 83.8, and 57.9% of methylation was observed in 10, 1, and 0.3 ng samples, respectively. This indicates that >50% of the genome was covered when the amount of starting DNA was decreased by 3 orders of magnitude.
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