Additional file 1 of Combined RT-qPCR and pyrosequencing of a Spike glycoprotein polybasic cleavage motif can uncover pediatric SARS-CoV-2 infections associated with heterogeneous presentation

Additional file 1: Table S1. Oligonucleotides used for RT-qPCR and pyrosequencing. Table S2. Observed cases of pediatric SARS-CoV-2 infections (overview). Figure S1. Results of semi-quantitative PCR for the performance comparison using GITC-purified RNA or stabilized raw specimens as RT-qPCR substrates. Four confirmed SARS-CoV-2-positive specimens (p1 to p4) were used in combination with primer pairs targeting Orf E, RdRP, Orf N and S-gene amplicons in singleplex qPCR reactions. From GITC-purified templates, amplicons with the correct size could be amplified from all specimens. Notably, we observed lower molecular weight byproducts for Orf E PCR. From stabilized raw specimens, in contrast, the Orf E amplicon was not amplified from none of the four samples. With varying band intensity, amplicons with correct sizes were amplified from stabilized raw samples for all cases for RdRP and S-gene targets and in some cases for the Orf N target. However, for unknown reasons, band intensities appeared less stable when compared with purified RNA samples. Moreover, we observed lower molecular weight byproducts in some cases. Figure S2. Results of semi-quantitative PCR of the SARS-CoV-2 S-gene amplicon. A. After 36 PCR cycles and subsequent agarose gel electrophoresis the specific 162 bp amplicon corresponding to the SARS-CoV-2 protein S-gene was visible (red arrow). The gel was loaded with 8 RT-qPCR samples from confirmed SARS-CoV-2-positive cases (p1 to p8) and 1 negative control (n1). The occasional weak appearance of PCR byproducts (see p1) seemed to correlate with relatively low viral (+)RNA load in the specimen. B. An excess of PCR cycles (40x) leads to enrichment of unspecific lower and higher molecular weight byproducts in all samples from confirmed positive samples (p1 to p9) as well as negative controls (n1 to n3). These byproducts severely impaired the successive pyrosequencing leading to ambiguous results. Figure S3. Results of a triplex PCR approach for the simultaneous detection of amplicon targets for RdRP, Orf E and Orf N. For comparison, 14 SARS-CoV-2-negative samples (n1 to n14) and 11 confirmed SARS-CoV-2-positive specimens were analyzed by agarose gel electrophoresis. The simultaneous use of primers for RdRP, Orf E and Orf N amplicons leads to enrichment of unspecific lower and higher molecular weight byproducts in all samples, confirmed positives as well as negatives. Remarkably, in the fraction of negative samples bands of approx. 160 bp appear frequently. Figure S4. Example of overlapping RT-qPCR curves. Curves of positive controls are red, negative specimens are green, water control is purple. Figure S5. Pyrograms for 5 pediatric SARS-CoV-2-positive cases. Below the pediatric cases one adult example is shown, where the (Spike) P681H substitution was detected. Figure S6. Presentation of a female toddler (age group 1-3 years) with a Multisystem Inflammatory Syndrome in Children (MIS-C)/Kawasaki-like syndrome associated with SARS-CoV-2 infection. A. Echocardiography revealed an enlargement of the left coronary artery (LCA) with a pericardial effusion. B. Flow cytometric characterization of the peripheral mononuclear blood cells resulted in normal ranges of CD3+ T, CD4+ T helper, CD19+ B, and CD16+CD56+ natural killer cells. Figure S7. Amplicon size tests by microvolume electrophoresis using the Agilent Bioanalyzer and position of sequencing primers on the 513 bp fragment. A. L: DNA size ladder, lanes 1-2: 162 bp S-gene (PBC) amplicon, 3-4: 513 bp S-gene (RBD) amplicon. B. Position of a sequencing primer for analyses of residues 484, 486, 489, 493 and 494 (red shaded) as well as residue 501 (underlined). C. Top: pyrogram showing N501 in one specimen; bottom: pyrogram showing N501Y in one specimen