Fig. 4

Deep targeted sequencing of SARS-CoV-2 by multiplex amplicon generation. a Amplicon targets. Six amplicons (dark line) ranging from 1 to 2.6 kb were used to target approximately 40% of the SARS-CoV-2 genome. The density of anchor 25-mers indicating high-sequence similarity across GISAID isolates is also shown. b Dynamic range of SARS-CoV-2 sequencing. Next-generation sequencing libraries were created from the SARS-CoV-2 amplicons as well as targeting the RPP30 control gene. Libraries consisted of serial dilutions (N = 8 replicates) of purified SARS-CoV-2 genomic RNA into total nucleic acid derived from a COVID-negative saliva sample. Reads aligning to either RPP30 or SARS-CoV-2 are shown, alongside with the log-log correlation to the number of input copies of virus in each sample. c Correlation with digital PCR. At each dilution used for sequencing, a portion of cDNA was used for digital PCR (N = 3 replicates), targeting the N1 gene. The corresponding concentration derived from digital PCR is shown alongside reads aligning to virus versus the RPP30 control (correlation coefficient = 0.996, p < 1.75e-7). d Limit of detection testing. A larger number of replicates (N = 32) was performed at 0, 1, and 2 input viral copies to assess the limit of detection of the assay. Shown are the reads aligning to both SARS-CoV-2 and the RPP30 control gene. Significance values (FDR-corrected) which test against the negative control (0 copies) are shown