Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations-Application to HIV-1 quasispecies.

Autor:	Westfall DH; Department of Microbiology, University of Washington School of Medicine, 960 Republican Street, Seattle, WA 98195-8070, USA., Deng W; Department of Microbiology, University of Washington School of Medicine, 960 Republican Street, Seattle, WA 98195-8070, USA., Pankow A; Department of Microbiology, University of Washington School of Medicine, 960 Republican Street, Seattle, WA 98195-8070, USA., Murrell H; Department of Pathology, Division of Medical Virology, University of Cape Town and National Health Laboratory Services, Observatory, Cape Town 7925, South Africa., Chen L; Department of Microbiology, University of Washington School of Medicine, 960 Republican Street, Seattle, WA 98195-8070, USA., Zhao H; Department of Microbiology, University of Washington School of Medicine, 960 Republican Street, Seattle, WA 98195-8070, USA., Williamson C; Department of Pathology, Division of Medical Virology, University of Cape Town and National Health Laboratory Services, Observatory, Cape Town 7925, South Africa., Rolland M; US Military HIV Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA.; The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., 6720A Rockledge Drive, Bethesda, MD 20817, USA., Murrell B; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Solnavägen 9, Stockholm 171 65, Sweden., Mullins JI; Department of Microbiology, University of Washington School of Medicine, 960 Republican Street, Seattle, WA 98195-8070, USA.; Department of Medicine, University of Washington School of Medicine, 960 Republican Street, Seattle, WA 98195-8070, USA.; Department of Global Health, University of Washington Schools of Medicine and Public Health, 960 Republican Street, Seattle, WA 98195-8070, USA.
Jazyk:	angličtina
Zdroj:	Virus evolution [Virus Evol] 2024 Mar 02; Vol. 10 (1), pp. veae019. Date of Electronic Publication: 2024 Mar 02 (Print Publication: 2024).
DOI:	10.1093/ve/veae019
Abstrakt:	Pathogen diversity resulting in quasispecies can enable persistence and adaptation to host defenses and therapies. However, accurate quasispecies characterization can be impeded by errors introduced during sample handling and sequencing, which can require extensive optimizations to overcome. We present complete laboratory and bioinformatics workflows to overcome many of these hurdles. The Pacific Biosciences single molecule real-time platform was used to sequence polymerase-chain reaction (PCR) amplicons derived from cDNA templates tagged with unique molecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR. The use of UMI allowed accurate template quantitation as well as removal of point mutations introduced during PCR and sequencing to produce a highly accurate consensus sequence from each template. Production of highly accurate sequences from the large datasets produced from SMRT-UMI sequencing is facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline). PORPIDpipeline automatically filters and parses circular consensus reads by sample, identifies and discards reads with UMIs likely created from PCR and sequencing errors, generates consensus sequences, checks for contamination within the dataset, and removes any sequence with evidence of PCR recombination, heteroduplex formation, or early cycle PCR errors. The optimized SMRT-UMI sequencing and PORPIDpipeline methods presented here represent a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus quasispecies in a virus transmitter-recipient pair of individuals. Competing Interests: The views expressed are those of the authors and should not be construed to represent the positions of the Department of Health and Human Services, the US Army, the Department of Defense, or the Henry M. Jackson Foundation for the Advancement of Military Medicine. (© The Author(s) 2024. Published by Oxford University Press.)
Databáze:	MEDLINE
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