Influenza Virus High-Throughput Influenza Sequencing Pipeline
- Sample collection
- Extraction of influenza virus genomic RNA
- RNA shipped to Wadsworth Center for full genome amplification
- Primer design in 96-well format Alternative pipeline: sample bar-coded with a unique sequence and multiplexed with other samples for 454 Sequencing.
- Amplification by RT-PCR or PCR of overlapping regions of each virus segment (1 virus isolate per 96-well plate; 4 plates per thermocycler)
- Sequencing of PCR amplicons using M13-universal primers in 384-well plate formats (4 virus isolates sequenced simultaneously)
- Assembly, tracking, and validation of influenza segments
- Quality assurance and submission to NCBI
