Ultra-low-input, tagmentation-based whole-genome bisulfite sequencing

Abstract

We have adapted transposase-based in vitro shotgun library construction ("tagmentation") for whole-genome bisulfite sequencing. This method, Tn5mC-seq, enables a >100-fold reduction in starting material relative to conventional protocols, such that we generate highly complex bisulfite sequencing libraries from as little as 10 ng of input DNA, and ample useful sequences from 1 ng of input DNA. We demonstrate Tn5mC-seq by sequencing the methylome of a human lymphoblastoid cell line to ∼8.6× high-quality coverage of each strand.

Figure 1.

The Tn5mC-seq method and resulting methylation profiles. (A) Tagmentation-based DNA-seq library construction. Genomic DNA is attacked by transposase homodimers loaded with synthetic, discontinuous oligos (yellow, purple) that allow for fragmentation and adaptor incorporation in a single step. Subsequent PCR appends outer flowcell-compatible primers (pink, green). (B) Tn5mC-seq library construction. Loaded transposase attacks genomic DNA with a single methylated adaptor (yellow). An oligo-replacement approach anneals a second methylated adaptor (purple), which is then subject to gap-repair. Bisulfite treatment then converts unmethylated cytosine to uracil (orange) followed by PCR to append outer flowcell-compatible primers (pink, green). Methylation is represented as black lollipops. (C) Coverage of cytosine positions genome-wide. More than 96% of Cs in all three contexts are covered at least once. Slight decrease in CpG coverage is due to reduced read alignment ability at regions with a high density of methylation. (D) Normalized methylated cytosine over total cytosine positions in 10-kb windows across chromosome 12 (blue and purple, left axis), and normalized methylated CpG over total CpG in 100-kb windows across chromosome 12 (green, right axis). (E) Normalized methylated CpG over total CpG residues at annotated genic loci. Promoter is defined as 2-kb region upstream of TSS. (F) Elevated CpG methylation levels in gene body (intron, exon) compared to intergenic regions.