{
    "abstract": "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 PCR amplicons derived from cDNA templates tagged with universal molecular identifiers (SMRT-UMI). Optimized laboratory protocols were developed through extensive testing of different sample preparation conditions to minimize between-template recombination during PCR and 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. Handling of the large datasets produced from SMRT-UMI sequencing was facilitated by a novel bioinformatic pipeline, Probabilistic Offspring Resolver for Primer IDs (PORPIDpipeline), that automatically filters and parses 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 or early cycle PCR errors, resulting in highly accurate sequence datasets. The optimized SMRT-UMI sequencing method presented here represents a highly adaptable and established starting point for accurate sequencing of diverse pathogens. These methods are illustrated through characterization of human immunodeficiency virus (HIV) quasispecies.",
    "authors": [
        {
            "affiliation": "Department of Microbiology, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195-8070 US.",
            "firstname": "Dylan H",
            "initials": "DH",
            "lastname": "Westfall"
        },
        {
            "affiliation": "Department of Microbiology, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195-8070 US.",
            "firstname": "Wenjie",
            "initials": "W",
            "lastname": "Deng"
        },
        {
            "affiliation": "Department of Microbiology, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195-8070 US.\nDepartment of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.",
            "firstname": "Alec",
            "initials": "A",
            "lastname": "Pankow"
        },
        {
            "affiliation": "Division of Medical Virology, Department of Pathology, University of Cape Town and National Health Laboratory Services, Cape Town, South Africa.",
            "firstname": "Hugh",
            "initials": "H",
            "lastname": "Murrell"
        },
        {
            "affiliation": "Department of Microbiology, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195-8070 US.",
            "firstname": "Lennie",
            "initials": "L",
            "lastname": "Chen"
        },
        {
            "affiliation": "Department of Microbiology, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195-8070 US.",
            "firstname": "Hong",
            "initials": "H",
            "lastname": "Zhao"
        },
        {
            "affiliation": "Division of Medical Virology, Department of Pathology, University of Cape Town and National Health Laboratory Services, Cape Town, South Africa.",
            "firstname": "Carolyn",
            "initials": "C",
            "lastname": "Williamson"
        },
        {
            "affiliation": "US Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland, 20910, USA.\nThe Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., Bethesda, Maryland, 20817, USA.",
            "firstname": "Morgane",
            "initials": "M",
            "lastname": "Rolland"
        },
        {
            "affiliation": "Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 17717 Stockholm, SE.",
            "firstname": "Ben",
            "initials": "B",
            "lastname": "Murrell"
        },
        {
            "affiliation": "Department of Microbiology, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195-8070 US.\nDepartment of Medicine, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195-8070 US.\nDepartment of Global Health, University of Washington Schools of Medicine and Public Health, Seattle, WA 98195-8070 US.",
            "firstname": "James I",
            "initials": "JI",
            "lastname": "Mullins"
        }
    ],
    "conclusions": null,
    "copyrights": null,
    "doi": "10.1101/2023.02.23.529831\n10.1371/journal.pgen.1008271\n10.1007/82_2015_453\n10.1128/JVI.64.2.864-872.1990\n10.1038/nrg.2016.49\n10.1007/978-1-4939-7471-9_12\n10.1093/nar/gky066\n10.1038/s41587-021-01108-x\n10.1093/hmg/ddy177\n10.1093/nar/26.7.1819\n10.1073/pnas.1110064108\n10.1038/nmeth.1778\n10.1073/pnas.1208715109\n10.1038/s41587-019-0217-9\n10.12688/f1000research.10571.2\n10.1371/journal.pone.0257521\n10.1016/j.ab.2006.10.009\n10.1093/nar/gkq543\n10.1093/ve/vew018\n10.1056/NEJMoa1508952\n10.1371/journal.ppat.1008179\n10.1089/aid.1997.13.737\n10.12688/f1000research.29032.2\n10.1101/gr.8.3.175\n10.1093/nar/gkz657\n10.1093/molbev/mst010",
    "journal": "bioRxiv : the preprint server for biology",
    "keywords": [],
    "methods": null,
    "publication_date": "2023-03-04",
    "pubmed_id": "36865215\n31622336\n26318138\n2296085\n27184599\n29423801\n29401301\n34750572\n29767702\n9512558\n22135472\n22101854\n22853953\n31406327\n28868132\n34597327\n17107651\n20571086\n29492273\n27192360\n32027734\n9171217\n3016298\n34035898\n9521921\n9521922\n31418021\n23329690",
    "results": null,
    "title": "Optimized SMRT-UMI protocol produces highly accurate sequence datasets from diverse populations - application to HIV-1 quasispecies.",
    "xml": "<Element 'PubmedArticle' at 0x788503ac7e70>"
}