De novo assembly of viral quasispecies using overlap graphs

Abstract

A viral quasispecies, the ensemble of viral strains populating an infected person, can be highly diverse. For optimal assessment of virulence, pathogenesis, and therapy selection, determining the haplotypes of the individual strains can play a key role. As many viruses are subject to high mutation and recombination rates, high-quality reference genomes are often not available at the time of a new disease outbreak. We present SAVAGE, a computational tool for reconstructing individual haplotypes of intra-host virus strains without the need for a high-quality reference genome. SAVAGE makes use of either FM-index-based data structures or ad hoc consensus reference sequence for constructing overlap graphs from patient sample data. In this overlap graph, nodes represent reads and/or contigs, while edges reflect that two reads/contigs, based on sound statistical considerations, represent identical haplotypic sequence. Following an iterative scheme, a new overlap assembly algorithm that is based on the enumeration of statistically well-calibrated groups of reads/contigs then efficiently reconstructs the individual haplotypes from this overlap graph. In benchmark experiments on simulated and on real deep-coverage data, SAVAGE drastically outperforms generic de novo assemblers as well as the only specialized de novo viral quasispecies assembler available so far. When run on ad hoc consensus reference sequence, SAVAGE performs very favorably in comparison with state-of-the-art reference genome-guided tools. We also apply SAVAGE on two deep-coverage samples of patients infected by the Zika and the hepatitis C virus, respectively, which sheds light on the genetic structures of the respective viral quasispecies.

Strain-aware assembly of genomes from mixed samples using flow variation graphs

“…This algorithm takes as input a data set of next-generation sequencing reads and a collection of strain-specific contigs; note that de novo assembly into strain-specific contigs can be performed using various tools (e.g. [16,19,26,27], depending on the application). The output of VG-flow consists of maximal length haplotypes along with relative abundance estimates for each of these sequences.…”

“…Existing viral quasispecies assemblers include widely evaluated tools like [12,13,14], as well a variety of methods introduced more recently [15,16,17,18,19,20]. These methods can be divided into two classes: reference-guided and reference-free (also referred to as de novo).…”

“…Especially at the time of a viral disease outbreak, an appropriate reference genome may not be available due to high mutation rates. However, most of the aforelisted tools are reference-guided; only [16], [17], and [19] present de novo approaches.…”

“…Among more generic assemblers, [26] has been shown to be capable of reconstructing individual haplotypes from mixed samples, up to a certain degree. This method was designed for bacterial genomes and scales well to human genomes, but is unable to reconstruct low-frequent haplotypes [16]. Haplotype-aware assembly of metagenomes is a big challenge, which tends to result in scattered genome fragments and missing strains [5].…”

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

“…Short-read secondgeneration sequencing technologies-such as Ion Torrent and Illumina-are restricted by read length (200-400 nt) (Hölzer and Marz 2017). For example, the use of highly fragmented viral RNAs considerably complicates the investigation of haplotypes (Nowak 1992;Baaijens et al 2017). Because the nested coronavirus mRNAs are almost identical to the original genome sequence, short-read data can usually not be unambiguously assigned to specific sg RNA species.…”