Enriched Methylomes of Low-input and Fragmented DNA Using Fragment Ligation EXclusive Methylation Sequencing (FLEXseq)

Jingru Yu; Lauren S. Ahmann; Yvette Y. Yao; Angus Martin Shaw Toland; Alicia Snowden; Chandler C. Ho; Netanel Loyfer; Tommy Kaplan; Hannes S. Vogel; Linlin Wang; Brooke E. Howitt; Brittany Holmes; Alarice Cheng-Yi Lowe; Wei Gu

doi:10.1093/nar/gkag385

GenomicsNucleic Acids ResearchApr 16, 2026

Peer-reviewed journal article

Enriched Methylomes of Low-input and Fragmented DNA Using Fragment Ligation EXclusive Methylation Sequencing (FLEXseq)

Jingru Yu iD, Lauren S. Ahmann iD, Yvette Y. Yao, Angus Martin Shaw Toland iD, Alicia Snowden, Chandler C. Ho iD, Netanel Loyfer iD, Tommy Kaplan iD, Hannes S. Vogel iD, Linlin Wang iD, Brooke E. Howitt iD, Brittany Holmes iD, Alarice Cheng-Yi Lowe iD, Wei Gu iD

Abstract

Methylome profiling is an emerging clinical tool for tumor classification and liquid biopsies. Here, we developed FLEXseq, a genome-wide methylation profiler that enriches and sequences the fragments of DNA flanking the CCGG motif. FLEXseq strongly correlates (Pearsons r = 0.97) with whole genome bisulfite sequencing (WGBS) while enriching 18-fold. To demonstrate the broad applicability of FLEXseq, we verified its usage across cells, body fluids, and formalin-fixed paraffin-embedded (FFPE) tissues. DNA dilutions down to 250 pg decreased CpG coverage, but bias in methylation remained low (Pearsons r [≥] 0.90) compared to a 10 ng input. FLEXseq offers a cost-efficient, base-pair resolution methylome with potential as a diagnostic tool for tissue and liquid biopsies.

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