Abstract
BackgroundWith rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols currently exist. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfite-based protocols and a new enzyme-based protocol. In addition, different input DNA quantities were compared for two kits compatible with a reduced starting quantity. In addition, we also present bioinformatic analysis pipelines for sequencing data from each of these library types.ResultsAn assortment of metrics were collected for each kit, including raw read statistics, library quality and uniformity metrics, cytosine retention, and CpG beta value consistency between technical replicates. Overall, the NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits performed quantitatively better than the other two protocols. In addition, the NEB and Swift kits performed well at low-input amounts, validating their utility in applications where DNA is the limiting factor.ResultsThe NEBNext Enzymatic Methyl-seq kit appeared to be the best option for whole-genome DNA methylation sequencing of high-quality DNA, closely followed by the Swift kit, which potentially works better for degraded samples. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Biosciences’s Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.
Highlights
With rapidly dropping sequencing cost, the popularity of whole-genome Deoxyribonucleic acid (DNA) methylation sequencing has been on the rise
The current goldstandard approach to examine genome-wide DNA methylation composition and differences is through chemical modification of unmethylated cytosines using sodium bisulfite [7]
The results presented are based on two snap-frozen primary patient fallopian tube samples
Summary
With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. We have performed 22 whole-genome DNA methylation sequencing experiments on snap frozen human samples, and extensively benchmarked common library preparation protocols for whole-genome DNA methylation sequencing, including three traditional bisulfitebased protocols and a new enzyme-based protocol. The current goldstandard approach to examine genome-wide DNA methylation composition and differences is through chemical modification of unmethylated cytosines using sodium bisulfite [7]. The end result yields stable genetic differences between methylated (C) and unmethylated cytosines (T), reflecting the underlying DNA methylation landscape, effectively turning the epigenetic difference into a genetic difference, which can be studied using conventional genome-scale methods, such as microarrays or sequencing. Various generations of bisulfite-based microarrays have been used to profile hundreds of thousands of human samples due to the low cost and easy, standardized data processing and analysis. With dropping sequencing cost in recent years, the popularity of sequencing-based methods has been on the rise [8]
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