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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.<\/jats:p>","DOI":"10.1093\/ve\/veae019","type":"journal-article","created":{"date-parts":[[2024,2,20]],"date-time":"2024-02-20T17:30:53Z","timestamp":1708450253000},"source":"Crossref","is-referenced-by-count":31,"title":["Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations\u2014Application to HIV-1 quasispecies"],"prefix":"10.1093","volume":"10","author":[{"given":"Dylan H","family":"Westfall","sequence":"first","affiliation":[{"name":"Department of Microbiology, University of Washington School of Medicine , 960 Republican Street, Seattle, WA 98195-8070, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenjie","family":"Deng","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington School of Medicine , 960 Republican Street, Seattle, WA 98195-8070, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alec","family":"Pankow","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington School of Medicine , 960 Republican Street, Seattle, WA 98195-8070, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hugh","family":"Murrell","sequence":"additional","affiliation":[{"name":"Department of Pathology, Division of Medical Virology, University of Cape Town and National Health Laboratory Services , Observatory, Cape Town 7925, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lennie","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington School of Medicine , 960 Republican Street, Seattle, WA 98195-8070, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington School of Medicine , 960 Republican Street, Seattle, WA 98195-8070, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolyn","family":"Williamson","sequence":"additional","affiliation":[{"name":"Department of Pathology, Division of Medical Virology, University of Cape Town and National Health Laboratory Services , Observatory, Cape Town 7925, South Africa"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3650-8490","authenticated-orcid":false,"given":"Morgane","family":"Rolland","sequence":"additional","affiliation":[{"name":"US Military HIV Research Program, Walter Reed Army Institute of Research , 503 Robert Grant Avenue, Silver Spring, MD 20910, USA"},{"name":"The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. , 6720A Rockledge Drive, Bethesda, MD 20817, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ben","family":"Murrell","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet , Solnav\u00e4gen 9, Stockholm 171 65, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4461-8158","authenticated-orcid":false,"given":"James I","family":"Mullins","sequence":"additional","affiliation":[{"name":"Department of Microbiology, University of Washington School of Medicine , 960 Republican Street, Seattle, WA 98195-8070, USA"},{"name":"Department of Medicine, University of Washington School of Medicine , 960 Republican Street, Seattle, WA 98195-8070, USA"},{"name":"Department of Global Health, University of Washington Schools of Medicine and Public Health , 960 Republican Street, Seattle, WA 98195-8070, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2024,3,2]]},"reference":[{"key":"2024051707304080800_R1","doi-asserted-by":"crossref","first-page":"284","DOI":"10.1128\/jvi.59.2.284-291.1986","article-title":"Production of Acquired Immunodeficiency Syndrome-Associated Retrovirus in Human and Nonhuman Cells Transfected with an Infectious Molecular Clone","volume":"59","author":"Adachi","year":"1986","journal-title":"Journal of Virology"},{"key":"2024051707304080800_R2","doi-asserted-by":"crossref","first-page":"2159","DOI":"10.1093\/nar\/gky066","article-title":"Single Molecule Real-Time (SMRT) Sequencing Comes of Age: Applications and Utilities for Medical Diagnostics","volume":"46","author":"Ardui","year":"2018","journal-title":"Nucleic Acids Research"},{"key":"2024051707304080800_R3","doi-asserted-by":"crossref","first-page":"84","DOI":"10.1016\/j.ab.2006.10.009","article-title":"Escherichia Coli DNA Polymerase III Epsilon Subunit Increases Moloney Murine Leukemia Virus Reverse Transcriptase Fidelity and Accuracy of RT-PCR Procedures","volume":"360","author":"Arezi","year":"2007","journal-title":"Analytical Biochemistry"},{"key":"2024051707304080800_R4","doi-asserted-by":"crossref","first-page":"1003","DOI":"10.1056\/NEJMoa2031738","article-title":"Two Randomized Trials of Neutralizing Antibodies to Prevent HIV-1 Acquisition","volume":"384","author":"Corey","year":"2021","journal-title":"New England Journal of Medicine"},{"key":"2024051707304080800_R5","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0182443","article-title":"Patterns and Rates of Viral Evolution in HIV-1 Subtype B Infected Females and Males","volume":"12","author":"Dapp","year":"2017","journal-title":"PLoS One"},{"key":"2024051707304080800_R6","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pone.0257521","article-title":"Sequencing DNA with Nanopores: Troubles and Biases","volume":"16","author":"Delahaye","year":"2021","journal-title":"PLoS One"},{"key":"2024051707304080800_R7","doi-asserted-by":"crossref","first-page":"405","DOI":"10.2144\/000113370","article-title":"DIVEIN: A Web Server to Analyze Phylogenies, Sequence Divergence, Diversity, and Informative Sites","volume":"48","author":"Deng","year":"2010","journal-title":"Biotechniques"},{"key":"2024051707304080800_R8","doi-asserted-by":"crossref","DOI":"10.1371\/journal.pgen.1008271","article-title":"Viral Quasispecies","volume":"15","author":"Domingo","year":"2019","journal-title":"PLOS Genetics"},{"key":"2024051707304080800_R9","first-page":"1","article-title":"What Is a Quasispecies? 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