Library-free methylation sequencing with bisulfite padlock probes

Abstract

Targeted quantification of DNA methylation allows for interrogation of the most informative loci across many samples quickly and cost-effectively. Here we report improved bisulfite padlock probes (BSPPs) with a design algorithm to generate efficient padlock probes, a library-free protocol that dramatically reduces sample-preparation cost and time and is compatible with automation, and an efficient bioinformatics pipeline to accurately obtain both methylation levels and genotypes from sequencing of bisulfite-converted DNA.

Figure 1

Schematic of library-free BSPP protocol. Each padlock probe has a common linker sequence flanked by two target-specific capturing arms (red) that anneal to bisulfite converted genomic DNA (black). The 3′ end is extended and ligated with the 5′ end to form circularized DNA. After removal of linear DNA, all circularized captured targets are PCR-amplified with barcoded primers and directly sequenced with an Illumina sequencing platform (GA II(x) or HiSeq). Amplicon size is 363 bp, which includes captured target (180 bp), capturing arms (55 bp), and amplification primers and adapters (128 bp). The inserts can be read through with paired-end 120 bp sequencing reads.