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

• 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
• Journal Title: Nucleic Acids Research
• Volume: Volume 43, Number 20
• Publisher: Oxford University Press (OUP) | 2015-11-16, Pages e129-e129
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1093/nar/gkv630
• Subjects: Biology, Molecular; Biology, Bioinformatics
• Keywords: Science & Technology; Life Sciences & Biomedicine; Biochemistry & Molecular Biology; IMMUNODEFICIENCY-VIRUS TYPE-1; DRUG-RESISTANCE; HETEROSEXUAL TRANSMISSION; DISCORDANT COUPLES; RATES; AMPLIFICATION; MUTATIONS; EVOLUTION; INFECTION; THERAPY

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.