Abstract
BackgroundCytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify types of cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group.ResultsEach sample is processed in multiple replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), enzymatic deamination method (EMSeq), targeted methylation sequencing (Illumina Methyl Capture EPIC), single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies, and 850k Illumina methylation arrays. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, bwa-meth, and BitMapperBS), we find overall high concordance between assays, but also differences in efficiency of read mapping, CpG capture, coverage, and platform performance, and variable performance across 26 microarray normalization algorithms.ConclusionsThe data provided herein can guide the use of these DNA reference materials in epigenomics research, as well as provide best practices for experimental design in future studies. By leveraging seven human cell lines that are designated as publicly available reference materials, these data can be used as a baseline to advance epigenomics research.
Highlights
DNA methylation plays a key role in the regulation of gene expression [1], disease onset [2], cellular development [1], age progression [3], and transposable element activity [4]
Study design and sequencing outputs We generated epigenomic data for seven well-characterized human cell lines (HG001HG007) that have been designated as reference materials for genomic benchmarking by the Genome in a Bottle (GIAB) Consortium [12]
Libraries for whole epigenome sequencing were prepared using a variety of common bisulfite and enzymatic conversion kits, including NEBNext Enzymatic Methyl-Seq, Swift Bio sciences Accel-NGS Methyl-Seq (MethylSeq), SPlinted Ligation Adapter Tagging (SPLAT), NuGEN TrueMethyl oxBS-Seq (TrueMethyl), and Illumina TruSeq DNA Methylation (TruSeq)
Summary
DNA methylation plays a key role in the regulation of gene expression [1], disease onset [2], cellular development [1], age progression [3], and transposable element activity [4]. The FDA’s Epigenomics Quality Control (EpiQC) Group presents DNA methylation sequence data across all seven GIAB reference cell lines, as well as a comparative analysis of targeted and genome-wide methylation protocols, to serve as a comprehensive resource for epigenetics research. We characterize the reproducibility and challenges of methylation estimation across the genome We further sequenced these cell lines using long-read technology on an Oxford Nanopore PromethION and here compare its performance alongside more common chemical/enzymatic conversion kits and short-read sequencing. We generated microarray data for these cell lines and provide guidelines for normalization of beta values, site filtration, and comparison to sequence data This reference dataset can act as a benchmarking resource and a reference point for future studies as epigenetics research becomes more widespread within the field of genomics. We present a multi-platform assessment and cross-validated resource for epigenetics research from the FDA’s Epigenomics Quality Control Group
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