Abstract
Influenza viruses still pose a serious threat to humans, and we have not yet been able to effectively predict future pandemic strains and prepare vaccines in advance. One of the main reasons is the high genetic diversity of influenza viruses. We do not know the individual clonotypes of a virus population because some are the majority and others make up only a small fraction of the population. First-generation (FGS) and next-generation sequencing (NGS) technologies have inherent limitations that are unable to resolve a minority clonotype’s information in the virus population. Third-generation sequencing (TGS) technologies with ultra-long reads have the potential to solve this problem but have a high error rate. Here, we evaluated emerging direct RNA sequencing and cDNA sequencing with the MinION platform and established a novel approach that combines the high accuracy of Illumina sequencing technology and long reads of nanopore sequencing technology to resolve both variants and clonotypes of influenza virus. Furthermore, a new program was written to eliminate the effect of nanopore sequencing errors for the analysis of the results. By using this pipeline, we identified 47 clonotypes in our experiment. We conclude that this approach can quickly discriminate the clonotypes of virus genes, allowing researchers to understand virus adaptation and evolution at the population level.
Highlights
Influenza A viruses (IAV) are negative-sense, single-stranded, segmented RNA viruses with characteristics of high genetic diversity [1,2,3]
Influenza virus RNA extracted from allantoic fluid was reverse-transcribed into viral cDNA and sequenced using the Illumina HiSeq platform, producing 4.5 GB of data
De novo assembly and subsequent BLAST annotation identified 12 IAV-related complete segments: PB2, PB1, PA, H5, H7, H9, NP, N2, N6, N9, M, and NS, and the raw data for next-generation sequencing (NGS) sequencing were submitted to the Sequence Read Archive (SRA) database
Summary
Influenza A viruses (IAV) are negative-sense, single-stranded, segmented RNA viruses with characteristics of high genetic diversity [1,2,3]. Wild birds are the natural reservoir of IAV, and bird migration can lead to IAV pandemics [4]. According to world health organization (WHO) estimates, the annual seasonal influenza can cause 5–10% of adults and 20–30% of children to become sick, with 3–5 million severe cases and 0.29–0.65 million deaths [5]. The live poultry trade has contributed to the continued presence of avian influenza virus in China [6,7]. As a big breeding country, IAV research has much significance for public health. The endemic prevalence of H5N6, H7N9 and
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