Long Single-Molecule Reads Can Resolve the Complexity of the Influenza Virus Composed of Rare, Closely Related Mutant Variants
As a result of a high rate of mutations and recombination events, an RNA-virus exists as a heterogeneous “swarm” of mutant variants. The long read length offered by single-molecule sequencing technologies allows each mutant variant to be sequenced in a single pass. However, high error rate limits the ability to reconstruct heterogeneous viral population composed of rare, related mutant variants. In this paper, we present 2SNV, a method able to tolerate the high error-rate of the single-molecule protocol and reconstruct mutant variants. 2SNV uses linkage between single nucleotide variations to efficiently distinguish them from read errors. To benchmark the sensitivity of 2SNV, we performed a single-molecule sequencing experiment on a sample containing a titrated level of known viral mutant variants. Our method is able to accurately reconstruct clone with frequency of 0.2 % and distinguish clones that differed in only two nucleotides distantly located on the genome. 2SNV outperforms existing methods for full-length viral mutant reconstruction. The open source implementation of 2SNV is freely available for download at http://alan.cs.gsu.edu/NGS/?q=content/2snv.
KeywordsSMRT reads RNA viral variants Single nucleotide variation
We would like to thank H. Hao for performing the PacBio sequencing at Johns Hopkins Deep Sequencing & Microarray Core Facility. A.A. was supported by GSU Molecular Basis of Disease Fellowship. S.M. and E.E were supported by National Science Foundation grants 0513612, 0731455, 0729049, 0916676, 1065276, 1302448 and 1320589, and National Institutes of Health grants K25-HL080079, U01-DA024417, P01- HL30568, P01-HL28481, R01-GM083198, R01-MH101782 and R01-ES022282. S.M. was supported in part by Institute for Quantitative & Computational Biosciences Fellowship, UCLA.
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