Assessment SARS-CoV-2 Amplicon Library Preparation and Genome Coverage.

a RT-PCR cycle thresholds predicting SARS-CoV-2 sequence read quality. Comparison of RT-PCR cycle thresholds for samples according to technical breadth and depth thresholds ((≥80% of SARS-CoV-2 genome covered at ≥100X read-depth; met thresholds, n = 140; did not meet thresholds n = 114). b SARS-CoV-2 full genome sequence depth comparisons. Among 140 sequences meeting our technical thresholds, the average coverage was 1,081 X (range 223 to 2,235 X); for the 114 sequences not meeting our technical thresholds, the average coverage was 71 X (range 0 to 1,281 X). Among all samples, average uniquely mapped read count was 292,430 reads (min = 21, max = 2,680,239). Among samples that pass >80% 100X coverage, the average uniquely mapped read count was 279,408 reads (min = 63,296, max = 2,680,239). Among samples that did not pass our cutoff, the average uniquely mapped read count was 175,895 reads (min = 21, max = 1,283,414). c Correlation between RT-PCR Ct value and average viral genome coverage; black circles identify samples that met the coverage and read-depth thresholds, and gray circles identify samples that did not meet these thresholds. d Correlation between detection of alternate alleles and RT-PCR Ct value where alternate allele frequency (AAF) was >5% (black circles >80?0x and open gray circles 50% (blue diamonds >80% 100x, open blue diamonds e Correlation between detection of alternate alleles and average SARS-CoV-2 genome coverage where alternate allele frequency (AAF) was >5% (black circles >80?0x and open gray circles 50% (blue diamonds >80% 100x, open blue diamonds f Correlation between the number of alternate alleles identified in the 140 >80% 100x samples and their collection dates—AAF >5% (black circles), or AAF >50% (blue diamonds). Significant correlation was observed between an increase in AAFs during the sample collection time-period (AAF >5%, Pearson R = 0.76112; AAF >50%, Pearson R = 0.89253; both P-values d-e identify samples that carried significantly more sequence variation compared to expectation (Sample 106–110 SNPs; Sample 110–132 SNPs). Green arrows in f link the position of the two samples with additional sequence variation at the AAF >50% and AAF >5% thresholds.