Figure 1. Library of IAV transcripts prepared for deep sequencing by Illumina HiSeq.

Human (A549) and mouse (M-1) cells were infected with the A/HongKong/1/1968 (H3N2) strain of IAV for four hours. The mRNA was extracted, an RNA oligonucleotide (yellow box) was ligated to the 5′ end, and the mRNAs were reverse-transcribed using a poly-dT primer. PCR amplification was performed on each IAV cDNA using the 5′ RACE primer and a gene-specific primer that annealed 20–40 nucleotides downstream of the ATG codon (highlighted in red). The region corresponding to the heterogeneous host-derived 5′ end of viral mRNA is shown in black. Four-nucleotide identifier tags (blue boxes) were added to each library for multiplex sequencing, and adapter sequences for sequencing (red boxes) were added onto the ends of the DNA.