Simultaneous and complete genome sequencing of influenza A and B with high coverage by Illumina MiSeq Platform

Abstract

Active global surveillance and characterization of influenza viruses are essential for better preparation against possible pandemic events. Obtaining comprehensive information about the influenza genome can improve our understanding of the evolution of influenza viruses and emergence of new strains, and improve the accuracy when designing preventive vaccines. This study investigated the use of deep sequencing by the next-generation sequencing (NGS) Illumina MiSeq Platform to obtain complete genome sequence information from influenza virus isolates. The influenza virus isolates were cultured from 6 respiratory acute clinical specimens collected in Thailand and Nepal. DNA libraries obtained from each viral isolate were mixed and all were sequenced simultaneously. Total information of 2.6 Gbases was obtained from a 455±14 K/mm2 density with 95.76% (8,571,655/8,950,724 clusters) of the clusters passing quality control (QC) filters. Approximately 93.7% of all sequences from Read1 and 83.5% from Read2 contained high quality sequences that were ≥Q30, a base calling QC score standard. Alignments analysis identified three seasonal influenza A H3N2 strains, one 2009 pandemic influenza A H1N1 strain and two influenza B strains. The nearly entire genomes of all six virus isolates yielded equal or greater than 600-fold sequence coverage depth. MiSeq Platform identified seasonal influenza A H3N2, 2009 pandemic influenza A H1N1and influenza B in the DNA library mixtures efficiently.

Molecular epidemiology of swine influenza A viruses in the Southeastern United States, highlights regional differences in circulating strains

“…The MiSeq Platform (Illumina, USA) was used for Next Generation Sequencing (NGS) and as described by Rutvisuttinunt and coworkers (Rutvisuttinunt et al, 2013). For sample preparation, cDNA synthesis was performed using Superscript III RT (Life Technologies, USA) combined with Uni-12 primer (AGCAAAAGCAGG), set at the thermocycler at 65°C for 5 minutes and then placed on ice for at least 1 minute.…”

Evaluation of Unbiased Next-Generation Sequencing of RNA (RNA-seq) as a Diagnostic Method in Influenza Virus-Positive Respiratory Samples

“…To our knowledge, this is the first comprehensive study to sequence complete genomes of seasonal influenza virus infections directly from diagnostic nucleic acid samples. Previous studies applying NGS with regard to influenza genome analysis were performed from smaller cohorts of pandemic influenza cases (30), from lung tissue (34), or after subculturing of the virus (35)(36)(37), which is laborious; in addition, dependent on the viral load, not all subculturing is successful. Other approaches applied amplicon sequencing approaches to selectively sequence all influenza genome segments (38).…”

Avian influenza H9N2 virus isolated from air samples in LPMs in Jiangxi, China

“…PCR products were purified by QIAGEN MinElute Reaction Cleanup Kit. One ng of DNA product was processed for NGS (next generation sequencing) sample preparation with the Illumina Nextera XT DNA Sample Preparation Kit (96 Samples), and sequencing was performed using a MiSeq v2 kit (500 cycles) to produce 2 × 250 paired-end reads (Illumina, San Diego, CA, USA) [15]. After automated cluster generation with MiSeq, the sequencing files were processed and genomic sequence reads were obtained.…”