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

Zhou, M.; Nabilsi, N. H.; Wang, A.; Gauthier, M.-P. L.; Murray, K. O.; Azari, H.; Owens, W. S.; Newman, J. R. B.; Pardo-Palacios, F. J.; Conesa, A.; Riva, A.; Clanton, T. L.; Reynolds, B. A.; Concannon, P.; Brant, J. O.; Bacher, R.; Kladde, M. P.

2022-11-13 cancer biology
10.1101/2022.11.08.515732 bioRxiv
Show abstract

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 by 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.

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