Fig. 4

Comparative characterization of pyrosequencing sensitivity and basecalling quality. Serial dilutions of the same clinical specimen were used for all tests. Top: targeted region and principle of S-gene fragment pyrosequencing. Limited by the used PCR primers, the maximum theoretical sequence length is 55 nt. Bottom left: resulting pyrograms from serial template dilutions used for RT-qPCR in singleplex reactions and subsequent pyrosequencing of the S-gene amplicons using the antisense single-strand (as defined by the biotinylated reverse primer [S_pbc-CoV-2-R_BIO]) are shown. From the undiluted singleplex reaction, we obtained the longest unbiased SARS-CoV-2-specific sequence fragment, which had an error-free length of 53 nt. Bottom right: resulting pyrograms from serial template dilutions used for RT-qPCR in duoplex reactions and subsequent pyrosequencing of the S-gene amplicons using the antisense single-strand (as defined by the biotinylated reverse primer [S_pbc-CoV-2-R_BIO]) are shown. From the undiluted duoplex reaction, we obtained a maximum unbiased SARS-CoV-2-specific sequence fragment of 44 nt in length. Bottom left/right: the associated C_T values are shown beside the degree of dilution. Lower template concentrations led to the occasional occurrence of miscalled bases (missing bases: red triangle; excess bases: blue triangle) and gradual convergence of signal and noise peaks. Whereas concomitantly, automated basecalling gradually failed to separate signal from noise, the SARS-CoV-2 specific sequence could be identified by manual inspection much longer