Fig. 6: Amplicon performance and mismatch analysis.
From: varVAMP: degenerate primer design for tiled full genome sequencing and qPCR

a For each sequencing result obtained via the virus specific primer scheme, the amplicon recovery (upper panel) and normalized coverage (lower panel) were calculated. Each color represents an individual amplicon for the respective schemes (see Table 1) tracked over different samples that are indicated on the x-axis. Amplicon recovery was calculated as the percentage of reference nucleotides covered at least 20-fold between the genomic start and stop position of the individual amplicons. For the normalized amplicon coverage, the mean coverage was calculated for each amplicon and normalized to the highest covered amplicon of each scheme (set to 100). b For each sequencing result, each primer binding region was analyzed for the number of mismatches not covered by any permutation of the corresponding primer sequence. Mutations were considered only if their variant frequency was ≥0.7. The primers were excluded from the analysis if any primer binding position was not covered at least 20-fold. c Dumbbell plot showing the pairwise identities of the newly generated fasta consensus sequences (blue dots) or the sequences of the varVAMP input MSA (dark gray dots) of each respective primer scheme. Light gray and red lines indicate the percent pairwise identity increase or decrease, respectively. Significance was calculated with a two-sided Welch’s t-test between the pairwise identities of newly produced sequences and alignment sequences for each respective virus. Exact p values are given above the minimum significance threshold of 0.001. (n.d. not determined as n < 3, n.s. not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001). d Percentage of off-target, unmapped reads for the respective schemes and samples (SARS-CoV-2: n = 14, BoDV-1: n = 3, HAV: n = 9, HEV cluster 2: n = 4, HEV cluster 4: n = 5, PV: n = 6, ratHEV: n = 4). Shown are mean ± STD.