Background: With rapidly dropping sequencing cost, the popularity of whole-genome DNA methylation sequencing has been on the rise. Multiple library preparation protocols exist, but a systematic evaluation and benchmarking of their performance against each other is currently lacking. We have performed 22 whole-genome DNA methylation sequencing experiments on fresh 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. Additionally, 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.
Results: An 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 kit performed quantitatively better than the other three protocols at two different DNA input amounts. Additionally, the results for the different input amounts were generally consistent across all metrics.
Conclusions: Based on these results, we recommend use of the NEBNext Enzymatic Methyl-seq kit for whole-genome DNA methylation sequencing. Further, a general bioinformatic pipeline is applicable across the four protocols, with the exception of extra trimming needed for the Swift Bioscience's Accel-NGS Methyl-Seq protocol to remove the Adaptase sequence.