Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing |
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| Authors: | Karen E. Ocwieja Scott Sherrill-Mix Rithun Mukherjee Rebecca Custers-Allen Patricia David Michael Brown Susana Wang Darren R. Link Jeff Olson Kevin Travers Eric Schadt Frederic D. Bushman |
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| Affiliation: | 1Perelman School of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, 2Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA, 3RainDance Technologies, Lexington, MA, 4Pacific Biosciences, Menlo Park, CA and 5Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York City, NY, USA |
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| Abstract: | Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-189.6 produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing. |
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