Long Single-Molecule Reads Can Resolve the Complexity of the Influenza Virus Composed of Rare, Closely Related Mutant Variants

Abstract

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 article, we present two single-nucleotide variants (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.

Long-read sequence and assembly of segmental duplications

“…First, our approach does not require PSVs to be predefined and, as such, can be applied to any genome assembly where long-read data of sufficient depth has been generated. A similar concept was recently applied to partition viral quasispecies 48 . Second, our validation results suggest that the paralog-specific assemblies are highly accurate (99.86%−99.99%).…”

Systematic benchmarking of omics computational tools

“…In these cases, instead of obtaining the golden standard, one can design a mock community (often referred to as a synthetic mock community) by combining titrated in vitro proportions of community elements. The most popular mock communities are prepared as mixtures of known microbial organisms 19,20 . When microbial organisms are closely related to similar sequences, such as intra-host RNA-virus populations, one should include closely related pairs and challenge computational methods with various frequency profiles 20–23 .…”

“…The most popular mock communities are prepared as mixtures of known microbial organisms 19,20 . When microbial organisms are closely related to similar sequences, such as intra-host RNA-virus populations, one should include closely related pairs and challenge computational methods with various frequency profiles 20–23 . Mock community gold standards offer numerous advantages, but they are artificial and typically comprised of a small number of members when compared with real communities; a smaller number of members increases the risk of oversimplifying reality.…”

Reconstruction of Microbial Haplotypes by Integration of Statistical and Physical Linkage in Scaffolding

“…2014 ; Pulido-Tamayo et al. 2015 ; Albanese and Donati 2017 ; Artyomenko et al. 2017 ; Posada-Cespedes et al.…”