Evaluation of whole-genome DNA methylation sequencing library preparation protocols

• Published in: Epigenetics & chromatin Vol. 14; no. 1; p. 28
• Main Authors: Morrison, Jacob, Koeman, Julie M, Johnson, Benjamin K, Foy, Kelly K, Beddows, Ian, Zhou, Wanding, Chesla, David W, Rossell, Larissa L, Siegwald, Emily J, Adams, Marie, Shen, Hui
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 19.06.2021; BioMed Central; BMC
• Subjects: Biological products industry; Biotechnology industry; Bisulfite; Comparative analysis; CpG islands; Cytosine; Deoxyribonucleic acid; DNA; DNA Methylation; DNA sequencing; Enzymatic methylation sequencing; Enzymes; Epigenetic inheritance; Epigenetics; Fallopian tube; Fallopian tubes; Gene Library; Genetic research; Genomes; Genomics; Humans; Methods; Methylation; Reproducibility; Sequence Analysis, DNA; Statistical analysis; Sulfites; Whole Genome Sequencing; Whole-genome bisulfite sequencing; United States; Whole-genome bisulfite sequencing; DNA methylation; Epigenetics; Fallopian tube; Enzymatic methylation sequencing
• ISSN: 1756-8935; 1756-8935
• DOI: 10.1186/s13072-021-00401-y

With 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. 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 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. The 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.