Enriched methylomes of low-input and fragmented DNA using fragment ligation EXclusive methylation sequencing

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.