A Targeted Bisulfite Sequencing Method Combining Microfluidics-Based Pcr With Next-Gen Sequencing

DNA methylation plays an important role in normal organismal development and in cellular differentiation in higher organisms. Changes in DNA methylation have been shown to correlate with disease risk, response to therapy and survival in a wide range of clinical conditions such as cancer and autoinflammatory diseases. Recent advances in next-generation sequencing and microarray technology have made it possible to map DNA methylation genome-wide, at a high resolution. However, these genomic assays tend to be costly, labor-intensive and impractical in the clinic. Thus, DNA methylation assays that measure a small number of genomic regions in large cohorts are needed for validating biomarker candidates discovered from genomic studies. For this reason, we have developed a targeted bisulfite sequencing platform for simultaneously measuring DNA methylation in multiple loci with multiple samples by combining microfluidics-based technology with next generation sequencing. First, post-bisulfite PCR primers for each target region were designed using our proprietary program Bisulfite Primer Seeker, and then experimentally validated using a standard genomic control. Primers which pass the validation were used for the subsequent target amplification. Target amplification were performed using Fluidigm Access Array 48.48 which allows parallel PCR reactions for 48 samples by 48 single-plex assays. An attractive aspect of this platform is that relatively small quantities of template are required (∼50 ng/sample). Assays can also be multiplexed to improve throughput. Furthermore, as with many targeted approaches, index or barcoding tags can be incorporated into the universal adapter regions of the PCR product enabling the pooling of samples before direct sequencing. Using this platform we have evaluated 9 genomic loci with 9 artificial mixed samples containing different methylation levels. Results showed the target bisulfite sequencing platform to be robust and reproducible. The methylation value between the technical replicates were highly correlated. Each of the amplicons was covered by a thousand or more reads, making the detection sensitivity reach down to the low methylation levels. Compared with other target bisulfite sequencing methods, our assay has several advantages. First, it uses relatively small amounts of gDNA as input, making it practical for most clinical samples. Secondly the workflow is simple, straightforward and can be automated. Finally, it avoids the complexity and difficulties of multiplex PCR by utilizing microfluidics-based single-plex PCR. All these features make it an ideal platform for DNA methylation biomarker validation. Citation Format: Emily Putnam, Lam Nguyen, Hunter Chung, Peisheng Shi, Xueguang Sun, Marc E. Van Eden, Xi-Yu Jia. A targeted bisulfite sequencing method combining microfluidics-based PCR with Next-Gen sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2956. doi:10.1158/1538-7445.AM2015-2956