Flap-enabled next-generation capture (FENGC): precision targeted single-molecule profiling of epigenetic heterogeneity, chromatin dynamics, and genetic variation

Targeted sequencing is an increasingly sought technology. Available methods, however, are often costly and yield high proportions of off-target reads. Here, we present FENGC, a scalable, multiplexed method in which target sequences are assembled into 5′ flaps for precise excision with flap endonuclease. Recovery of length-matched sequences, amplification with universal primers, and exonucleolytic removal of non-targeted genomic regions mitigate amplification biases and consistently yield ≥ 80% on-target sequencing. Furthermore, optimized sequential reagent addition and purifications minimize sample loss and facilitate rapid processing of sub microgram quantities of DNA for detection of genetic variants and DNA methylation. Treatment of cultured human glioblastoma cells and primary murine monocytes with GC methyltransferase followed by FENGC and high-coverage enzymatic methyl sequencing provides single-molecule, long-read detection of differential endogenous CG methylation, dynamic nucleosome repositioning, and transcription factor binding. FENGC provides a versatile and cost-effective platform for targeted sequence enrichment for analysis of genetic and/or epigenetic heterogeneity. ### Competing Interest Statement Mingqi Zhou, Nancy Nabilsi, and Michael Kladde are inventors (assignee, University of Florida) of pending patent application (PCT/US2022/020624, filed March 16, 2022) for the FENGC technology.