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Título: | Flap-enabled next-generation capture (FENGC): precision targeted single-molecule profiling of epigenetic heterogeneity, chromatin dynamics, and genetic variation |
Autor: | Zhou, Mingqi; Nabilsi, Nancy H.; Wang, Anqi; Gauthier, Marie-Pierre L.; Murray, Kevin O.; Azari, Hassan; Owens, William S.; Newman, Jeremy R. B.; Pardo-Palacios, Francisco J.; Conesa, Ana CSIC ORCID; Riva, Alberto; Clanton, Thomas L.; Reynolds, Brent A.; Concannon, Patrick; Brant, Jason O.; Bacher, Rhonda; Kladde, Michael P. | Fecha de publicación: | 2022 | Editor: | Cold Spring Harbor Laboratory Press | Citación: | bioRxiv: 515732 (2022) | Resumen: | 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. | Versión del editor: | https://doi.org/10.1101/2022.11.08.515732 | URI: | http://hdl.handle.net/10261/330797 | DOI: | 10.1101/2022.11.08.515732 |
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