Since the primers only anneal to a total of 4.5% of the viral genome, the chance that viral mutations will disrupt primer annealing is lower. This results in a lowered chance of amplicon dropouts.
The Ct value for the amplicon sequencing reaction is an important metric since the higher the Ct, the lower the number of viral genomes in the sample. Samples with less viral genomic material pose difficulty for primer annealing
In our experiment, contrived samples were prepared by mixing inactivated SARS-CoV-2 viral material into a negative background matrix of a negative nasopharyngeal (NP) swab in viral transport medium (VTM), with two samples per Ct value. Results show that
when the SARS-CoV-2 Midnight Amplicon Panel and xGen DNA Library Prep Kit EZ are combined, the recommended Ct value is <32 which has been shown to sequence 97% of the SARS-CoV-2 genome with sufficient depth to identify variants (Table 1).
Table 1. SARS-CoV-2 Midnight Amplicon Panel and xGen DNA Library Prep Kit EZ metrics.
|Recommended Ct value
||Contiguous amplicons (count)
|<32 ||97% ||29 ||30–28,985 |