Empirical validation of viral quasispecies assembly algorithms: state-of-the-art and challenges

Abstract

Next generation sequencing (NGS) is superseding Sanger technology for analysing intra-host viral populations, in terms of genome length and resolution. We introduce two new empirical validation data sets and test the available viral population assembly software. Two intra-host viral population 'quasispecies' samples (type-1 human immunodeficiency and hepatitis C virus) were Sanger-sequenced, and plasmid clone mixtures at controlled proportions were shotgun-sequenced using Roche's 454 sequencing platform. The performance of different assemblers was compared in terms of phylogenetic clustering and recombination with the Sanger clones. Phylogenetic clustering showed that all assemblers captured a proportion of the most divergent lineages, but none were able to provide a high precision/recall tradeoff. Estimated variant frequencies mildly correlated with the original. Given the limitations of currently available algorithms identified by our empirical validation, the development and exploitation of additional data sets is needed, in order to establish an efficient framework for viral population reconstruction using NGS.

Figure 1. Phylogenetic trees (upper panels) and networks (lower panels) of the original Sanger sequences for the HCV (left panels) and HIV-1 (right panels) data sets.

Neighbor-joining and neighbor-net algorithms were run on optimized models of evolution, over 500 bootstrap runs. Node labels show bootstrap percentages. Numbers after the labels represent variant prevalence (%).

Figure 2. Evolutionary history inferred by neighbor-joining, using an optimized nucleotide substitution model, that compares HCV variants reconstructed by each quasispecies assembler with the original Sanger clones; trees are rooted using the mapping reference sequence.

Panels (a), (b), (c), and (d) show Geneious™ de novo, PredictHaplo, QuRe and ShoRAH, respectively. Node numbers represent% bootstrap replicates (of 500) ≥ 75%. Bullets represent variants at a frequency ≥ 5%, and triangles those < 5% (not available for PredictHaplo, shown by squares). Blue color indicates Sanger isolates, and red reconstructed variants.

Figure 3. Evolutionary history inferred by neighbor-joining, using an optimized nucleotide substitution model, that compares HIV-1 variants reconstructed by each quasispecies assembler with the original Sanger clones; trees are rooted using the mapping reference sequence.

Panels (a), (b), (c), and (d) show Geneious™ de novo, PredictHaplo, QuRe and ShoRAH, respectively. Node numbers represent% bootstrap replicates (of 500) ≥ 75%. Bullets represent variants at a frequency ≥ 5%, and triangles those < 5% (not available for PredictHaplo, shown by squares). Blue color indicates Sanger isolates, and red reconstructed variants.

Figure 4. Phylogenetic trees comparing together all reconstructed variants from different assemblers with the original Sanger sequences for the HCV and HIV-1 experiments (left and right panels, respectively).

Trees have been inferred using neighbor-joining on an optimized model of evolution, rooted on the mapping reference sequence, performing 500 bootstraps (nodes with ≥ 75% bootstrap support are shown). For ease of read, only the 30 highest-frequency variants from ShoRAH have been included.