Epigenetic profiling is an emerging clinical tool for tumor profiling and liquid biopsies. Here, we developed Fragment Ligation EXclusive methylation sequencing (FLEXseq), a genome-wide methylation profiler that enriches and sequences the DNA fragments flanking CCGG motifs to cover a broad range of regulatory regions. FLEXseq strongly correlates with whole genome bisulfite sequencing (WGBS; Pearson's r = 0.97) yet requires only three- to seven-fold less sequencing depth than whole genome approaches to cover cell type-specific markers and 4- to 20-fold less sequencing to cover key regulatory regions like promoters. DNA dilutions down to 250 pg decreased CpG coverage, but bias in methylation remained low (Pearson's r ≥ 0.90). To demonstrate the broad applicability of FLEXseq, including highly fragmented DNA found in clinical specimens, we verified its usage across cells, body fluids, and formalin-fixed paraffin-embedded (FFPE) tissues. Across 106 cerebrospinal fluids, a specimen type challenged by low-input and fragmented DNA, FLEXseq enabled cell type deconvolution to distinguish between different tumor types and negative controls with an accuracy of 97%. FLEXseq offers a cost-efficient, single-nucleotide resolution approach to profile the methylome even with fragmented, low-input DNA.
Yu et al. (Thu,) studied this question.