Systematic evaluation of library preparation methods and sequencing platforms for high-throughput whole genome bisulfite sequencing

Abstract

Whole genome bisulfite sequencing (WGBS), with its ability to interrogate methylation status at single CpG site resolution epigenome-wide, is a powerful technique for use in molecular experiments. Here, we aim to advance strategies for accurate and efficient WGBS for application in future large-scale epidemiological studies. We systematically compared the performance of three WGBS library preparation methods with low DNA input requirement (Swift Biosciences Accel-NGS, Illumina TruSeq and QIAGEN QIAseq) on two state-of-the-art sequencing platforms (Illumina NovaSeq and HiSeq X), and also assessed concordance between data generated by WGBS and methylation arrays. Swift achieved the highest proportion of CpG sites assayed and effective coverage at 26x (P < 0.001). TruSeq suffered from the highest proportion of PCR duplicates, while QIAseq failed to deliver across all quality metrics. There was little difference in performance between NovaSeq and HiSeq X, with the exception of higher read duplication rate on the NovaSeq (P < 0.05), likely attributable to the higher cluster densities on its flow cells. Systematic biases exist between WGBS and methylation arrays, with lower precision observed for WGBS across the range of depths investigated. To achieve a level of precision broadly comparable to the methylation array, a minimum coverage of 100x is recommended.

Third-generation sequencing-based mapping and visualization of single nucleotide polymorphism, meiotic recombination, illegitimate mutation and repeat-induced point mutation

“…The genomic DNA was then subjected to library preparation using the Accel-NGS Methyl-Seq DNA library kit (catalog # 30024; Swift Bioscience, USA). The libraries were sequenced using a NextSeq 500 sequencing system (Illumina, USA) as described previously ( 23 ). BatMeth2, an open source software program ( https://github.com/GuoliangLi-HZAU/BatMeth2/ ) ( 24 ), was used for genome-wide DNA methylation calling.…”

Evaluation of Whole-Exome Enrichment Solutions: Lessons from the High-End of the Short-Read Sequencing Scale

“…On one side, biological duplication occurs randomly when two identical fragments were produced by DNA fragmentation, whereas the more problematic duplicates are generated in the PCR step [ 40 ] during library preparation. Optical and ExAmp duplicates are also generated during the sequencing process [ 41 , 42 ]. Optical duplicates are defined if the distance between the flow cell coordinates leading to two reads is within a 2500-pixel distance set by Picard’s OPTICAL_DUPLICATE_PIXEL_DISTANCE parameter.…”

Systemic Investigation of Promoter-wide Methylome and Genome Variations in Gout

“…EPIC BeadChip array has been validated as a very reliable genomic platform for determining DNA methylation patterns in the human genome [ 60 ]. Although experiments with next-generation sequencing approaches might offer more comprehensive coverage of genome-wide CpG sites [ 61 ], the more expensive cost, larger DNA requirement, significantly increased sample processing time [ 61 ], and the amount of sequencing required to achieve precision similar to that obtained in a methylation array [ 62 ] all have made next-generation sequencing approaches difficult to implement for most research budgets. Currently, most epigenome-wide studies (~99%) have been exclusively performed on methylation arrays [ 62 ].…”