Fig. 4

PCR amplification prior to sequencing leads to similar overall measurements of genetic diversity. a We compared our PrimalSeq that enriches for specific virus sequences to the current ‘gold standard’ for measuring intrahost genetic diversity, metagenomics; and we compared sequencing the amplicons using the Illumina and Oxford Nanopore platforms. The schematic outlines the general workflow for all approaches. b We sequenced our mixed Zika virus population (1000 virus RNA copies) containing 10% virus # 2 (Expected) in triplicate using both approaches and platforms to compare the accuracy of measuring known intrahost single-nucleotide variants (iSNVs). We only analyzed regions of the Zika virus #1 and #2 genomes (Fig. 1a) that were perfect matches to the PCR primer sequences, leaving 61 iSNV sites. Data shown as mean and range of triplicate tests. c We combined the frequency measurements for each iSNV site and replicate (n = 183) to compare the accuracy between the two approaches and platforms. Dashed line shows the expected true iSNV frequencies at 10%. Data shown as means and standard deviations. The mean frequencies were not significantly different (ns, Welch’s t test, p > 0.05), but the variances were not equal (*, Levene’s test, p < 0.05). d We analyzed the frequency of false positive iSNVs > 3% (cutoff determined in Fig. 1c) from each sequencing method and technical replicate (“A, B, C”) from 4173 sites that are expected to be true negatives. From our metagenomics and PCR-Illumina sequencing data, the same false positive iSNVs > 3% frequency are not found in multiple technical replicates, however, many are found in the PCR-Nanopore replicates (see Fig. 5). Dashed line shows the iSNV cutoff at 3%