title: Multiplexed highly-accurate DNA sequencing of closely-related HIV-1 variants using continuous long reads from single molecule, real-time sequencing.
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.
authors.affiliation: Emory Vaccine Center, Emory University, Atlanta, GA, 30329, USA.
authors.forename: Dario A
authors.initials: DA
authors.lastname: Dilernia
authors.affiliation: Emory Vaccine Center, Emory University, Atlanta, GA, 30329, USA.
authors.forename: Jung-Ting
authors.initials: JT
authors.lastname: Chien
authors.affiliation: Emory Vaccine Center, Emory University, Atlanta, GA, 30329, USA.
authors.forename: Daniela C
authors.initials: DC
authors.lastname: Monaco
authors.affiliation: Pacific Biosciences Inc., Menlo Park, CA, 94025, USA.
authors.forename: Michael P S
authors.initials: MP
authors.lastname: Brown
authors.affiliation: Emory Vaccine Center, Emory University, Atlanta, GA, 30329, USA.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Ende
authors.affiliation: Emory Vaccine Center, Emory University, Atlanta, GA, 30329, USA.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Deymier
authors.affiliation: Emory Vaccine Center, Emory University, Atlanta, GA, 30329, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Yue
authors.affiliation: Pacific Biosciences Inc., Menlo Park, CA, 94025, USA.
authors.forename: Ellen E
authors.initials: EE
authors.lastname: Paxinos
authors.affiliation: Pathology and Laboratory Medicine, Emory University, Atlanta, 30322, GA.
authors.forename: Susan
authors.initials: S
authors.lastname: Allen
authors.affiliation: University of Texas Health Science Center, San Antonio, TX, 78229, USA.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Tirado-Ramos
authors.affiliation: Emory Vaccine Center, Emory University, Atlanta, GA, 30329, USA Pathology and Laboratory Medicine, Emory University, Atlanta, 30322, GA ehunte4@emory.edu.
authors.forename: Eric
authors.initials: E
authors.lastname: Hunter
mesh_terms: D000465:Algorithms; D016000:Cluster Analysis; D014644:Genetic Variation; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*