An essential feature of each diagnostic PCR or qPCR assay is to detect as many genetic variants of a given virus or bacterial taxon as possible. A crucial step in such a screening
assay design is a proper oligonucleotide selection. Therefore, identification of the evolutionarily most conserved regions of the targeted virus genome is of utmost importance.
However, this crucial step is frequently underestimated in the literature. We set up a simple workflow how to align, process, and evaluate a huge set of homologous nucleotide
sequences in order to reveal the variability at each particular position. All of the steps were performed by using an ordinary desktop computer without the need for extensive
mathematical or computational skills. The workflow was demonstrated on aligning more than 15.000 avian influenza A virus (IAV) M segment nucleotide sequences. The whole
procedure takes a few hours and usually do not exceed a half working day in dependence on the internet connection speed and server occupancy respectively.