Beyond the consensus: dissecting within-host viral population diversity of foot-and-mouth disease virus by using next-generation genome sequencing - PubMed (original) (raw)

Beyond the consensus: dissecting within-host viral population diversity of foot-and-mouth disease virus by using next-generation genome sequencing

Caroline F Wright et al. J Virol. 2011 Mar.

Abstract

The diverse sequences of viral populations within individual hosts are the starting material for selection and subsequent evolution of RNA viruses such as foot-and-mouth disease virus (FMDV). Using next-generation sequencing (NGS) performed on a Genome Analyzer platform (Illumina), this study compared the viral populations within two bovine epithelial samples (foot lesions) from a single animal with the inoculum used to initiate experimental infection. Genomic sequences were determined in duplicate sequencing runs, and the consensus sequence of the inoculum determined by NGS was identical to that previously determined using the Sanger method. However, NGS revealed the fine polymorphic substructure of the viral population, from nucleotide variants present at just below 50% frequency to those present at fractions of 1%. Some of the higher-frequency polymorphisms identified encoded changes within codons associated with heparan sulfate binding and were present in both foot lesions, revealing intermediate stages in the evolution of a tissue culture-adapted virus replicating within a mammalian host. We identified 2,622, 1,434, and 1,703 polymorphisms in the inoculum and in the two foot lesions, respectively: most of the substitutions occurred in only a small fraction of the population and represented the progeny from recent cellular replication prior to onset of any selective pressures. We estimated the upper limit for the genome-wide mutation rate of the virus within a cell to be 7.8 × 10(-4) per nucleotide. The greater depth of detection achieved by NGS demonstrates that this method is a powerful and valuable tool for the dissection of FMDV populations within hosts.

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Figures

FIG. 1.

FIG. 1.

Coverage of the reference genome obtained with filtered, trimmed reads. (A) First data set (run), (B) second data set (run). The three samples (inoculum, front left foot, and back right foot) received generous coverage from both runs, while fluctuations were higher for the first data set. Average coverage values were ×4,873 (inoculum), ×8,665 (FLF sample), and ×6,594 (BRF sample) for the first data set and ×16,827 (inoculum), ×11,924 (FLF sample), and ×15,945 (BRF sample) for the second data set. At the top of the figure, the sequenced fraction of the genome (nt 368 to 8176) is represented, together with the position of the polyprotein.

FIG. 2.

FIG. 2.

Frequencies of mismatches (first data set) obtained by aligning the reads to the reference genome. (A) Inoculum; (B) FLF sample; (C) BRF sample. The average mismatch frequency lay around 0.1% for all three samples. At a few sites, the mismatch frequency was higher; as expected, the number of these peaks was larger for the FLF and BRF samples than for the inoculum. A small fraction of sites showed perfect agreement of all reads with the reference genome (mismatch frequency = 0).

FIG. 3.

FIG. 3.

Frequencies of mismatches (second data set) obtained by aligning the reads to the reference genome. (A) Inoculum; (B) FLF sample; (C) BRF sample. The second data set had a higher level of coverage than the first one, with a smaller fraction of sites with no mismatches. The average mismatch frequency is very similar to that of the first data set.

FIG. 4.

FIG. 4.

Correlations of polymorphism frequencies in viral populations. Correlations were computed between the two runs (first row) and within each run (second and third rows). The Spearman rank correlation ρ is indicated for each pair of data sets. Only data for qualitatively validated SSPs receiving coverage above ×100 in both runs are shown. The correlation coefficients between the two runs for the inoculum and the FLF sample are similar, while they are lower for the BRF sample. The remaining panels show that the first run was more correlated than the second run.

FIG. 5.

FIG. 5.

Variability in viral populations. Frequency distributions of the weighted averaged mismatch frequencies between the two runs are shown for the three samples (the ordinate represents the frequencies of sites showing each fraction of mismatches). Solid lines, all sites receiving minimum coverage of ×100 in both runs (7,755 sites for inoculum, 7,730 for FLF sample, and 7,710 sites for BRF sample); dashed lines, sites receiving coverage of ×100 or more in both runs and classified as validated SSPs (2,622 sites for inoculum, 1,434 for FLF sample, and 1,703 for BRF sample). All lines show similar trends: a small fraction of the sites (<1%) display no variability for both runs, most of the sites show a very small amount of polymorphism in the viral population (between 0.01% and 1%), and a very small fraction of the sites (0.14% for inoculum, 0.22% for FLF sample, and 0.39% for BRF sample) present variation at a level above 1%.

FIG. A1.

FIG. A1.

Average errors in reads, computed with base qualities. (A) First data set; (B) second data set. The average error increased greatly towards the ends of the reads (solid lines). The second data set was less noisy. Different filtering strategies were tested, and only reads whose average error was below a threshold θ value were accepted. More-stringent thresholds decreased the errors in the reads (small dashed, dotted, dotted-dashed, and dashed lines). The insets show the fractions of reads retained after the filtering process (using a threshold θ value of 0.2%), including 66% of the reads in the first data set and 95% of the reads in the second data set.

FIG. A2.

FIG. A2.

Distribution of mismatches to the reference genome on the reads after alignment. (Left) First data set; (right) second data set. The curves are largely flat, indicating an even distribution of mismatches over the reads, apart for a mild increase towards the edges of the reads, possibly due to reads containing insertions and deletions. We kept only data coming from the flat region of the curve, i.e., nucleotides 5 to 45 in each aligned read.

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