Dataset Information

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.

SUBMITTER: Dilernia DA

PROVIDER: S-EPMC4787755 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Publications

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

Dilernia Dario A DA Chien Jung-Ting JT Monaco Daniela C DC Brown Michael P S MP Ende Zachary Z Deymier Martin J MJ Yue Ling L Paxinos Ellen E EE Allen Susan S Tirado-Ramos Alfredo A Hunter Eric E

Nucleic acids research 20150622 20

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 Continuo ...[more]

PMID: 26101252

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Project description:BackgroundTargeted resequencing with high-throughput sequencing (HTS) platforms can be used to efficiently interrogate the genomes of large numbers of individuals. A critical issue for research and applications using HTS data, especially from long-read platforms, is error in base calling arising from technological limits and bioinformatic algorithms. We found that the community standard long amplicon analysis (LAA) module from Pacific Biosciences is prone to substantial bioinformatic errors that raise concerns about findings based on this pipeline, prompting the need for a new method.ResultsA single molecule real-time (SMRT) sequencing-error correction and assembly pipeline, C3S-LAA, was developed for libraries of pooled amplicons. By uniquely leveraging the structure of SMRT sequence data (comprised of multiple low quality subreads from which higher quality circular consensus sequences are formed) to cluster raw reads, C3S-LAA produced accurate consensus sequences and assemblies of overlapping amplicons from single sample and multiplexed libraries. In contrast, despite read depths in excess of 100X per amplicon, the standard long amplicon analysis module from Pacific Biosciences generated unexpected numbers of amplicon sequences with substantial inaccuracies in the consensus sequences. A bootstrap analysis showed that the C3S-LAA pipeline per se was effective at removing bioinformatic sources of error, but in rare cases a read depth of nearly 400X was not sufficient to overcome minor but systematic errors inherent to amplification or sequencing.ConclusionsC3S-LAA uses a divide and conquer processing algorithm for SMRT amplicon-sequence data that generates accurate consensus sequences and local sequence assemblies. Solving the confounding bioinformatic source of error in LAA allowed for the identification of limited instances of errors due to DNA amplification or sequencing of homopolymeric nucleotide tracts. For research and development in genomics, C3S-LAA allows meaningful conclusions and biological inferences to be made from accurately polished sequence output.

Dataset Information

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

Publications

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

Similar Datasets

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