Cost-effective targeted methyl-seq using an xGen ™ Custom Hyb Capture Panel and the xGen Methyl-Seq DNA Library Prep Kit identifies DNA methylation in low input samples consistent with WGBS

Performing comprehensive methylation profiling, such as whole genome bisulfite sequencing (WGBS), is a gold standard for target discovery. Once target methylation signatures are known, a more cost effective approach is desirable to increase the number of samples analyzed. This can be achieved with targeted methylation sequencing that is limited to methylation signatures of epigenetic interest. Using the xGen Methyl-Seq DNA Library Prep Kit paired with an xGen Custom Hyb Capture Panel to create target-enriched NGS libraries has been shown to improve coverage for desired regions at a significantly lower cost than WGBS without impacting data quality. The complete workflow supports methylation detection, high-performance target enrichment, and enables methylation detection in low input samples that is equivalent to data generated by WGBS. Human methylated and non-methylated DNA standards (Zymo Research) from genomic DNA (gDNA) were combined in varying ratios to create a range of samples representing 0%, 15%, 50%, 85%, and 100% genome-wide methylation levels. Samples were sheared to 300 bp using a Covaris S220 and bisulfite conversion was performed in triplicate using the EZ DNA Methylation-Gold Kit (Zymo Research) starting with 50 ng from each of the five representative mixes. All bisulfite-treated material was carried forward to create xGen Methyl-Seq DNA libraries using modifications outlined in Appendix A of the xGen Methyl-Seq DNA Library protocol. A second set of libraries were prepared as described above using 5, 10, or 25 ng of cfDNA reference material (RM) (Horizon HD833) into bisulfite conversion and without any mechanical shearing. A methylation-specific xGen Custom Hyb Panel was designed to enrich for five genes associated with methylation biomarkers, covering a total region of 128 kb. Hybridization capture was performed on all libraries following the xGen Hybridization Capture protocol, hybridizing three libraries per capture for 16 hours. Sequencing was performed on a NextSeq ™ (Illumina) platform using 2x75 bp reads with 10% phiX spike-in. For all analyses, 10 bp were trimmed from each end due to the low complexity tail added during the Adaptase step (see the application note, Tail trimming for better data, for more information). Each captured library was sub-sampled to 2 million total reads, and one WGBS from cfDNA RM was sub-sampled to 860 million total reads. Targeted sequencing data including alignment to the human genome (hg38) and methylation analyses were performed using Bismark (v0.22.3) and Picard (v2.18.9). For all samples, conversion efficiency was consistently ≥99% (data not shown). The xGen Methyl-Seq DNA Library Prep Kit is a robust and flexible solution driven by Adaptase technology, a library preparation chemistry which directly tails and performs adapter ligation to single-stranded bisulfite treated DNA fragments. In traditional bisulfite sequencing, the library adapters are added prior to bisulfite conversion, which can result in fragmented non-functional library molecules. A unique single-strand strategy efficiently creates NGS libraries that allow researchers to measure methylation levels from a wide range of sample input amounts and over a wide range of methylation levels. Libraries enriched using a methyl-specific xGen Custom Hyb Panel show methylation signatures that are highly correlated with whole genome sequencing data from the same sample, and therefore, targeted methyl-seq provides a cost-effective way to assess methylation of key targets associated with different disease states. A unique xGen Custom Hyb Panel design targeting both DNA strands post-conversion resulted in efficient capture of a complete range of simulated methylation levels and allows for deeper sequencing of regions of interest in comparison to whole-genome sequencing. Together, the xGen Methyl-Seq Library Prep Kit and an xGen Custom Hyb Panel create a meaningful tool for accelerating focused epigenetic research.