(740a) Ultrasensitive Microfluidic Assay for Genome-Wide DNA Methylation Analysis and Precision Medicine

Epigenetics refer to any process that alters gene activities without changing the DNA sequence. Previous studies have demonstrated that epigenetic regulation, such as DNA methylation, plays a critical role in many biological procedures, such as embryo development and learning process. Genome-wide study of dynamics of DNA methylation at single-base resolution provides a comprehensive understanding of methylome configuration. Bisulfite sequencing is regarded as the gold-standard technology for analyzing DNA methylation. This technology is based on the different consequences after cytosine and 5-methycytosine (5-mC) treated with sodium bisulfite. However, conventional protocols required more than 300 ng DNA due to the significant sample loss during bisulfite treatment (up to 98% ^{1, 2}). This limits its application for studying precious samples and understanding the underlying heterogeneity of clinic samples. To broaden the applications of bisulfite sequencing, it is necessary to develop ultrasensitive tools for epigenomic study, particularly DNA methylation analysis. We reported the first microfluidic DNA methylation assay at genome-wide scale and single-base resolution with >3 orders of magnitude higher sensitivity than conventional protocols.

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.

References

1.         M. Ehrich, S. Zoll, S. Sur and D. van den Boom, Nucleic Acids Res., 2007, 35, e29-e29.

2.         C. Grunau, S. J. Clark and A. Rosenthal, Nucleic Acids Res., 2001, 29, E65-65.

3.         S. Ma, D. N. Loufakis, Z. Cao, Y. Chang, L. E. K. Achenie and C. Lu, Lab Chip, 2014, 14, 2905-2909.