Fig. 7

PrimalSeq can be used to measure intrahost variants from a variety of sample types. a We sequenced technical duplicates of Zika virus populations (1000 virus RNA copies each) to identify intrahost single-nucleotide variants (iSNVs) > 3% within each sample. In vitro and in vivo samples were generated using Zika virus strain PRVABC59 (isolated from Puerto Rico, 2015) during infection of Ae. aegypti Aag2 cells (derived from embryos), human HeLa cells (derived from cervical epithelial cells), Ae. aegypti mosquitoes (orally infected), and Indian origin rhesus macaques (subcutaneously infected). For the in vitro and in vivo samples, where the reference population sequence is known, the iSNV frequencies were calculated by change in frequency from pre- to post-infection. Field Zika virus samples from pooled Ae. aegypti and human clinical samples were collected from Florida during the 2016 Zika virus outbreak. b Culex mosquitoes and dead American crows were collected from San Diego County, CA, during 2015 to sequence West Nile virus from field samples (10,000 virus RNA copies each). The iSNV frequencies from the field samples are the minor allele frequencies (maximum frequency = 0.5) because the reference virus sequence was not known. For both (a and b), analysis was limited to regions of the genome with > 400× coverage depth in the protein coding sequence and we masked amplicons with primer mismatches from our analysis (gray regions) for direct comparisons of intrahost genetic diversity