Table 1.
Summary of RT-PCR and sequencing results from no-template control reactions with multiple reagent production sources of the CDC 2019-Novel Coronavirus (2019-nCoV) real-time reverse transcriptase RT-PCR diagnostic panel.
Fig 1.
Next generation sequence data from products of the EUA Kit N1.
34% of reads generated from the EUA-kit N1 NTC reaction with false-positive reactivity mapped to the SARS-CoV-2 Wuhan-Hu-1 reference sequence (highlighted sequence at top) but contained four distinguishing SNPs (highlighted in the mapped reads) consistent with a contaminating template synthesized at CDC around the same time as the kit production (S1a Fig). The N1 primer and probe binding sites are annotated above the reference sequence.
Table 2.
Sequencing results of RT-PCR products demonstrated the source of false reactivity in N1 and N3 components.
Fig 2.
a. The NGS read length distribution (insert sizes after adapter and quality trimming) from the EUA-kit N3 false positive product demonstrating three prominent populations of molecules at 36 bp, 46 bp, and 58 bp in length. All three populations were identified as originating from the N3 assay components and comprise oligonucleotide duplex or triplex molecules (Fig 2b). b. Representative NGS reads from the three most common populations of the EUA-kit N3 false-positive RT-PCR reaction products (Fig 2a). N3 primers and probes are mapped below each read (reverse primer shown in reverse-complement). The 58 bp product and 36 bp products involve the probe and therefore would have generated fluorescence in the absence of template during the reaction. These same populations of products were generated from all three sets of primers and probes (pre-EUA, EUA-kit, commercial vendor).