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Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing.

ABSTRACT: Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. 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.We first compared the accuracy and sensitivity of both sequencers using plasmid DNA and different ratios of wild type and mutant plasmid. Illumina MiSeq sequencing reads were one and a half times more accurate than those of the Ion Torrent PGM. The majority of sequencing errors were substitutions on the Illumina MiSeq and insertions and deletions, mostly in homopolymer regions, on the Ion Torrent PGM. 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. We also optimized an RT-PCR protocol to obtain uniform coverage of all eight genomic RNA segments. The sequencing reads obtained with both sequencers could successfully be assembled de novo into the segmented influenza virus genome. 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. This threshold exceeds the background error rate resulting from the RT-PCR reaction and the sequencing method. Most of the variants in the PR8 virus genome were present in hemagglutinin, and these mutations were detected by both sequencers.Our approach underlines the power and limitations of two commonly used next-generation sequencers for the analysis of influenza virus gene diversity. 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. 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.

SUBMITTER: Van den Hoecke S

PROVIDER: S-EPMC4342091 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing.

Van den Hoecke Silvie S Verhelst Judith J Vuylsteke Marnik M Saelens Xavier X

BMC genomics 20150214

<h4>Background</h4>Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. 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.<h4>Re ...[more]

PMID: 25758772

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Project description:The influenza A(H1N1)pdm09 virus emerged in April 2009 with an unusual incidence of severe disease and mortality, and currently circulates as a seasonal influenza virus. Previous studies using consensus viral genome sequencing data have overlooked the viral genomic and phenotypic diversity. Next-generation sequencing (NGS) may instead be used to characterize viral populations in an unbiased manner and to measure within-host genetic diversity. In this study, we used NGS analysis to investigate the within-host genetic diversity of influenza A(H1N1)pdm09 virus in the upper and lower respiratory samples from nine patients who were admitted to the intensive care unit (ICU). A total of 47 amino acid substitution positions were found to differ between the upper and lower respiratory tract samples from all patients. However, the D222G/N substitution in hemagglutinin (HA) protein was the only amino acid substitution common to multiple patients. Furthermore, the substitution was detected only in the six samples from the lower respiratory tract. Therefore, it is important to investigate influenza A(H1N1)pdm09 virus populations using multiple paired samples from the upper and lower respiratory tract to avoid overlooking potentially important substitutions, especially in patients with severe disease.IMPORTANCE The D222G/N substitution in the hemagglutinin (HA) protein of influenza A(H1N1)pdm09 virus has been reported to be associated with disease severity and mortality in numerous previous studies. In the present study, 75% of lower respiratory samples contained heterogeneous influenza populations that carried different amino acids at position 222 of the HA protein, whereas all upper respiratory samples only contained the wild-type 222D. These results suggest the influenza A(H1N1)pdm09 virus has diversified inside the host owing to differences in tissue specificity. In this study, the within-host genetic diversity of influenza A(H1N1)pdm09 virus was investigated for the first time using next-generation sequencing analysis of the viral whole-genome in samples extracted from the upper and lower respiratory tracts of patients with severe disease.

Project description:Approximately 36.7 million people were living with the human immunodeficiency virus (HIV) at the end of 2016 according to UNAIDS, representing a global prevalence rate of 0.8%. In Brazil, an HIV prevalence of 0.24% has been estimated, which represents approximately 830,000 individuals living with the virus. As a touristic and commercial hub in Latin America, Brazil harbors an elevated HIV genetic variability, further contributed by the selective pressure exerted by the host immune system and by antiretroviral treatment. Through the progress of the next-generation sequencing (NGS) techniques, it has been possible to expand the study of HIV genetic diversity, evolutionary, and epidemic processes, allowing the generation of HIV complete or near full-length genomes (NFLG) and improving the characterization of intra- and interhost diversity of viral populations. Greater sensitivity in the detection of viral recombinant forms represents one of the major improvements associated with this development. It is possible to identify unique or circulating recombinant forms using the near full-length viral genomes with increasing accuracy. It also permits the characterization of multiple viral infections within individual hosts. Previous Brazilian studies using NGS to analyze HIV diversity were able to identify several distinct unique and circulating recombinant forms and evidenced dual infections. These data unveiled unprecedented high rates of viral recombination and highlighted that novel recombinants are continually arising in the Brazilian epidemic. In the pooled analysis depicted in this report, HIV subtypes have been determined from HIV-positive patients in five states of Brazil with some of the highest HIV prevalence, three in the Southeast (Rio de Janeiro, São Paulo, and Minas Gerais), one in the Northeast (Pernambuco) and one in the South (Rio Grande do Sul). Combined data analysis showed a significant prevalence of recombinant forms (29%; 101/350), and a similar 26% when only NFLGs were considered. Moreover, the analysis was able to evidence the occurrence of multiple infections in some individuals. Our data highlight the great HIV genetic diversity found in Brazil and unveils a more accurate scenario of the HIV evolutionary dynamics in the region.

Dataset Information

Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing.

Publications

Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing.

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