| Autor: |
Mingqi Zhou, Nancy H. Nabilsi, Anqi Wang, Marie-Pierre L. Gauthier, Kevin O. Murray, Hassan Azari, William S. Owens, Jeremy R. B. Newman, Francisco J. Pardo-Palacios, Ana Conesa, Alberto Riva, Thomas L. Clanton, Brent A. Reynolds, Patrick Concannon, Jason O. Brant, Rhonda Bacher, Michael P. Kladde |
| Rok vydání: |
2022 |
| DOI: |
10.1101/2022.11.08.515732 |
| Popis: |
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. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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