Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing

• Type of Material: Article
• Authors: Dario A. Dilernia, Emory University; Jung-Ting Chien, Emory University; Daniela Monaco, Emory University; Michael P.S. Brown, Pacific Biosciences Inc.; Zachary Ende, Emory University; Martin J. Deymier, Emory University; Ling Yue, Emory University; Ellen E. Paxinos, Pacific Biosciences Inc.; Susan Allen, Emory University; Alfredo Tirado-Ramos, University of Texas Health Science Center; Eric Hunter, Emory University
• Language: English
• Date: 2015-11-16
• Publisher: Oxford University Press (OUP)
• Publication Version: Final Published Version
• Copyright Statement: © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1093/nar/gkv630
• Title of Journal or Parent Work: Nucleic Acids Research
• ISSN: 0305-1048
• Volume: 43
• Issue: 20
• Start Page: e129
• End Page: e129
• Grant/Funding Information: amfAR [Mathilde Krim Fellowship in Basic Biomedical Research; 108672-5-RKGN to D.D.]; Institute of Allergy and Infectious Diseases at the National Institutes of Health [R01 AI64060 and R37 AI51231 to E.H.]; Virology Core at the Emory Center for AIDS Research [P30 AI050409] (in part); Yerkes National Primate Research Center base grant [2P51RR000165-51] through the National Center for Research Resources [P51RR165]; Office of Research Infrastructure Programs/OD [P51OD11132]; Action Cycling Fellowships (to D. D., D. M. and M. D.); E.H. is a Georgia Eminent Scholar.; Funding for open access charge: Institute of Allergy and Infectious Diseases at the National Institutes of Health [R01 AI64060 and R37 AI51231 to E.H.]; amfAR [108672-55-RKGN to D.D.].
• Keywords: DRUG-RESISTANCE; RATES; EVOLUTION; INFECTION; THERAPY; MUTATIONS; IMMUNODEFICIENCY-VIRUS TYPE-1; Life Sciences & Biomedicine; HETEROSEXUAL TRANSMISSION; DISCORDANT COUPLES; AMPLIFICATION; Biochemistry & Molecular Biology; Science & Technology
• Research Categories: Biology, Molecular; Biology, Bioinformatics

Abstract

Single Molecule, Real-Time (SMRT®) Sequencing (Pacific Biosciences, Menlo Park, CA, USA) provides the longest continuous DNA sequencing reads currently available. However, the relatively high error rate in the raw read data requires novel analysis methods to deconvolute sequences derived from complex samples. Here, we present a workflow of novel computer algorithms able to reconstruct viral variant genomes present in mixtures with an accuracy of >QV50. This approach relies exclusively on Continuous Long Reads (CLR), which are the raw reads generated during SMRT Sequencing. We successfully implement this workflow for simultaneous sequencing of mixtures containing up to forty different >9 kb HIV-1 full genomes. This was achieved using a single SMRT Cell for each mixture and desktop computing power. This novel approach opens the possibility of solving complex sequencing tasks that currently lack a solution.