Abstract 4232: Increased sensitivity in whole-genome bisulfite sequencing (WGBS): A novel “post-bisulfite conversion” library construction method from sub-nanogram DNA inputs.

Abstract

Abstract Genome-wide analysis of 5-methylcytosines is possible with whole-genome bisulfite sequencing (WGBS), where unmethylated cytosine residues are converted to uracil. However, a major challenge in WGBS is the degradation of DNA that occurs during bisulfite conversion under conditions required for complete conversion. Typically, ∼90% of input DNA is degraded and thus, is especially problematic with limited starting amounts of DNA. Additionally, regions that are rich in unmethylated cytosines are more sensitive to strand breaks. As a consequence, a majority of DNA fragments contained in di-tagged NGS DNA libraries treated with bisulfite “post-library construction” can be rendered inactive due to strand breaks in the DNA sequence flanked by the adapter sequences. These mono-tagged templates are then excluded during library enrichment resulting in incomplete coverage and bias when performing whole genome bisulfite sequencing. Here, we describe a novel “post-bisulfite conversion” library construction method for preparing NGS libraries from genomic DNA prior to the addition of one or both adapters. This “post-bisulfite conversion” library construction method uses the resulting untagged or mono-tagged single-stranded DNA as template for the subsequent addition of adapter sequences required for NGS. Thus, single-stranded DNA fragments independent of size and position of strand breaks remain as viable templates for library construction, eliminating the loss of fragments and the selection bias associated with a “post-library construction” bisulfite conversion strategy. This novel “post-bisulfite conversion” library construction method exhibits high diversity, increased efficiency and sensitivity (500 picograms human genomic DNA input), and improved coverage required for WGBS. Citation Format: Roy Sooknanan, John Hitchen, Nick Caruccio. Increased sensitivity in whole-genome bisulfite sequencing (WGBS): A novel “post-bisulfite conversion” library construction method from sub-nanogram DNA inputs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4232. doi:10.1158/1538-7445.AM2013-4232