Whole Genome Sequencing of Influenza Virus

RNAs were extracted from the A/Perth/16/2009 WT and resistant viruses and subjected to reverse transcription and PCR amplification using the PathAmp FluA Reagents (Thermo Fisher). A fraction of the PCR products were resolved and visualized on 1% agarose gel to ensure the presence of all 8 influenza genome segments (PB2, PB1, PA, HA, NP, NA, M, NS) at the expected sizes (2.31, 2.27, 2.12, 1.70, 1.54, 1.41, 0.98, 0.84 kb). The remaining PCR products were purified using the DNA Clean & Concentrator (Zymo Research), and contracted to SeqWright (GE Healthcare) for library preparation and whole genome sequencing with Illumina paired-end technology (2x100bp) on the Illumina Hiseq platform (Illumina). The genomic alignment software GSNAP was used for all FASTQ sequence alignment [48 (link)]. GenBank sequences of A/Perth/16/2009 (Accession No. KJ609203—KJ609210) were used as the reference sequences for alignment (S1 Table). Sequence analysis was carried out by Genentech in-house viral bioinformatics pipeline based on R and Bioconductor packages: GenomicRanges [49 (link)], GenomicAlignments [49 (link)], VariantTools [50 ], gmapR [51 ]. Only base-calls with Q-score ≥ 30 were tallied for variant calling. Amino acid substitutions with respect to the reference sequences were determined at a frequency threshold of 50%.