This approach revealed a mutation rate of approximately 1.5 × 10 −5 per nucleotide per infectious cycle. In other words, viral genomes that are fit enough to generate plaques were sequenced. The mutation rate of influenza A viruses has been traditionally determined by sequencing different cDNA clones obtained from multiple plaques descending from a plaque-purified influenza A virus. Mutations that are introduced during replication are tolerated because they are neutral for virus fitness in a particular environment, rapidly lost because they reduce fitness, or expanded because they are advantageous. Replication of the RNA genome of influenza viruses is associated with a relatively high mutation rate (2.3 × 10 −5) because the viral RNA-dependent RNA polymerase lacks 3′-5′-exonuclease activity and therefore has no proof-reading function. These viruses represent three of the five genera of the Orthomyxoviridae family, which is characterized by enveloped viruses that have a segmented, single-stranded, negative sense RNA genome. It is caused by influenza A and B viruses and occasionally by influenza C virus. Influenza is an acute and highly contagious viral disease of the respiratory tract in humans. This situation favors the emergence of fit mutant viruses that escape the herd immunity induced by infection with parental viruses or by vaccination. Human influenza viruses represent a prototypical example of rapid virus evolution facilitated by error-prone genome replication combined with the selection pressure imposed by host immune responses. Biologically, the quasispecies is the level at which selection takes place. A viral quasispecies is defined as a proliferating population of non-identical but closely related viral genomes in a mutation-prone environment subjected to continuous competition and selection. Later, the term was also used to describe an RNA virus population consisting of a mixture of related genomes. The term ‘quasispecies theory’ was first introduced by Manfred Eigen as a theoretical model to study molecular evolution by mutation and selection in self-reproducing macromolecules. Viral genomic diversity is well captured in the term ‘quasispecies’. Furthermore, viruses in general and RNA viruses in particular have a huge genetic diversity, which is the driving force of their evolutionary success. Viruses outnumber all other known life forms on earth. The data analysis pipeline that we propose here will also help to standardize variant calling in small RNA genomes based on next-generation sequencing data. We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time. Our approach underlines the power and limitations of two commonly used next-generation sequencers for the analysis of influenza virus gene diversity. Most of the variants in the PR8 virus genome were present in hemagglutinin, and these mutations were detected by both sequencers. This threshold exceeds the background error rate resulting from the RT-PCR reaction and the sequencing method. After mapping of the reads to the reference genome, we found that the detection limit for reliable recognition of variants in the viral genome required a frequency of 0.5% or higher. The sequencing reads obtained with both sequencers could successfully be assembled de novo into the segmented influenza virus genome. We also optimized an RT-PCR protocol to obtain uniform coverage of all eight genomic RNA segments. To evaluate the suitability of the two techniques for determining the genome diversity of influenza A virus, we generated plasmid-derived PR8 virus and grew this virus in vitro. The majority of sequencing errors were substitutions on the Illumina MiSeq and insertions and deletions, mostly in homopolymer regions, on the Ion Torrent PGM. Illumina MiSeq sequencing reads were one and a half times more accurate than those of the Ion Torrent PGM. We first compared the accuracy and sensitivity of both sequencers using plasmid DNA and different ratios of wild type and mutant plasmid. We compared the suitability of two benchtop next-generation sequencers for whole genome influenza A quasispecies analysis: the Illumina MiSeq sequencing-by-synthesis and the Ion Torrent PGM semiconductor sequencing technique. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies.
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