Fig. 7: In-depth analysis of a rhinovirus C detection by mNGS that was discrepant with RT-PCR.

A A heat map generated from SURPI+ analysis shows 12 reads aligning to rhinovirus C from a single sample, excluding the possibility of cross-contamination. Each column denotes a clinical sample, while each row corresponds to a taxonomic identification at the species, genus, or family level. The asterisks refer to “declassification” of reads from one level to the next higher taxonomic level (for example, from species to genus). B A coverage map shows that the 12 reads span the genome of the most closely matched rhinovirus C genome in the reference database identified by SURPI+ (accession number MG148341.1) without overlap, with coverage of 19% of the ~7000 base pair (bp) genome. C Several mismatches in the primer and probe sequences from published RT-PCR assays targeting the 5’-untranslated region (5’-UTR) are observed when compared to the viral mNGS reads, providing a likely explanation for the discrepant mNGS and RT-PCR results. The four assays are labeled 1 through 4 and correspond to Lu et al.²⁹ (1), Tapparel et al.⁴⁷ (2), Gunson et al.⁴⁸ (3), and Steininger et al.⁴⁹ (4). The mismatched nucleotides are highlighted with a background colour. Note that the assay from Steininger, et al. does not include a probe. Abbreviations: FP forward primer, Pr probe, RP reverse primer.