Fig. 1

Overview of the workflow for in silico evaluation of RT-qPCR methods. A two-step BLAST approach is used to first recover the genomic regions targeted by the RT-qPCR method under investigation in every analysed genome, after which the annealing regions for the primers and probe are extracted. Hybridisation properties of the primer/probe-template pair are then investigated by means of a set of selection criteria that mimic the PCR reaction: mismatch percentage (a maximum of 20% of bases can be mismatched in the primer/probe), alignment length (a minimum primer/probe alignment length of 80% is required), and number of mismatched bases in the 3′ end region of primers (either one or no single mismatch is allowed in the last five bases of this region). Threshold values for these selection criteria were set in accordance with previous observations documented in the literature (see Discussion). Genomes are considered as detected only if all three criteria are met, and are otherwise classified as not detected. Unknown cases represent genomes where the targeted genomic region cannot be extracted, because it either is not present or alternatively incomplete and located at the beginning or end of the genomic sequence. See Methods for an extended description of the workflow