Abstract
BackgroundThe advent of next-generation sequencing technologies and the resultant lower costs of sequencing have enabled production of massive amounts of data, including the generation of full genome sequences of pathogens. However, the small genome size of the influenza virus arguably justifies the use of the more conventional Sanger sequencing technology which is still currently more readily available in most diagnostic laboratories.ResultsWe present a simplified Sanger-based genome sequencing method for sequencing the influenza A/H3N2 virus in a large-scale format. The entire genome sequencing was completed with 19 reverse transcription-polymerase chain reactions (RT-PCRs) and 39 sequencing reactions. This method was tested on 15 native clinical samples and 15 culture isolates, respectively, collected between 2009 and 2011. The 15 native clinical samples registered quantification cycle values ranging from 21.0 to 30.56, which were equivalent to 2.4×103–1.4×106 viral copies/µL of RNA extract. All the PCR-amplified products were sequenced directly without PCR product purification. Notably, high quality sequencing data up to 700 bp were generated for all the samples tested. The completed sequence covered 408,810 nucleotides in total, with 13,627 nucleotides per genome, attaining 100% coding completeness. Of all the bases produced, an average of 89.49% were Phred quality value 40 (QV40) bases (representing an accuracy of circa one miscall for every 10,000 bases) or higher, and an average of 93.46% were QV30 bases (one miscall every 1000 bases) or higher.ConclusionsThis sequencing protocol has been shown to be cost-effective and less labor-intensive in obtaining full influenza genomes. The constant high quality of sequences generated imparts confidence in extending the application of this non-purified amplicon sequencing approach to other gene sequencing assays, with appropriate use of suitably designed primers.
Highlights
In recent years, advances in sequencing techniques have enabled an increasing number of research studies based on the genome-wide sequences of the influenza viruses [1,2,3,4,5,6], rather than relying solely on an individual gene that may preclude more comprehensive gene signatures [7,8]
Since the large number of influenza genome sequences deposited by Ghedin et al [4] and the initiation of the Influenza Genome Sequencing Project in 2005 [9], the deposition of complete human influenza A virus genomes by other groups has increased exponentially
We describe a whole genome sequencing method for seasonal influenza A/H3N2, with modifications of the normal sequencing protocol that reduces the number of processing steps, but still constantly produces a high quality sequence read of up to 700 bp
Summary
Advances in sequencing techniques have enabled an increasing number of research studies based on the genome-wide sequences of the influenza viruses [1,2,3,4,5,6], rather than relying solely on an individual gene that may preclude more comprehensive gene signatures [7,8]. The eight RNA segments encode for (in the order of the segment numbers one to eight): viral RNA polymerase basic 2 (PB2, 2341 nt), polymerase basic 1 (PB1, 2341 nt), polymerase acidic (PA, 2233 nt), hemagglutinin (HA, 1762 nt), nucleoprotein (NP, 1567 nt), neuraminidase (NA, 1466 nt), matrix (M1, 1027 nt), and nonstructural (NS1, 890 nt) protein. Apart from these proteins, alternatively spliced mRNAs of the seventh segment (M1) and the eighth segment (NS1) allow translation of two additional proteins, namely, the ion channel matrix protein (M2) and nuclear export/nonstructural protein (NEP/NS2), respectively. The small genome size of the influenza virus arguably justifies the use of the more conventional Sanger sequencing technology which is still currently more readily available in most diagnostic laboratories
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