The Seattle Flu Study: a multi-arm community-based prospective study protocol for assessing influenza prevalence, transmission, and genomic epidemiology

Helen Y. Chu; Michael Boeckh; Janet A. Englund; Michael Famulare; Barry R. Lutz; Deborah A Nickerson; Mark J. Rieder; Lea M Starita; Amanda Adler; Elisabeth Brandstetter; Chris D. Frazar; Peter D. Han; Reena K. Gularti; James Hadfield; Michael L. Jackson; Anahita Kiavand; Louise E. Kimball; Kirsten Lacombe; Jennifer Logue; Victoria Lyon; Kira L. Newman; Thomas R. Sibley; Monica L. Zigman Suschsland; Caitlin Wolf; Jay Shendure; Trevor Bedford

Laboratory methods All study respiratory specimens are aliquoted in triplicate and barcoded using a unique identifier that can be linked back to the participant or site of collection. Samples were frozen at -80˚C until thawed for extraction ( Figure 2 ). Total nucleic acids are extracted from 200 µl of UTM using Magna Pure 96 small total nucleic acids extraction kit (Roche). Extracted nucleic acids are screened for the presence of respiratory pathogens by TaqMan RT-PCR on the OpenArray platform (Thermo) ( Table 2 ). Specimens that test positive for influenza by RT-PCR are then sequenced using a modified oligo capture protocol. Total RNA is converted to cDNA and sequencing libraries are constructed using the Illumina TruSeq RNA Library Prep for Enrichment kit, as outlined by the manufacturer (Illumina, San Diego, CA). Library samples are ranked by TaqMan CRT value, and 24 samples with similar CRT values are pooled prior to oligo capture. CRT values are further used to determine the viral load of each sample and inform the fraction of each capture pool that a given sample represents. Each pool of flu-positive samples is hybridized overnight to a custom pool of capture oligonucleotides following the manufacturer's recommended protocol (Twist Bioscience, San Francisco, CA). Final libraries are sequenced on the Illumina Miseq, NextSeq or NovaSeq platform with paired end 150bp reads.