In silico evaluation and selection of the best 16S rRNA gene primers for use in next-generation sequencing to detect oral bacteria and archaea

Table 3 Selected primer pairs for detecting oral archaea in different amplicon-length categories

Species coverage was estimated as the number of species with at least one match in an ASV divided by the number of species included in the database. Our bacterial and archaeal databases contained 769 and 194 species, respectively, each of which had between one and 4000 ASVs. The location of the first and last nucleotides of each primer within each sequence with a match was calculated and the mode values for these positions were determined. If there was more than one mode for a position, we chose the one closest to the mean position value. As all the sequences in the two databases were aligned with the 16S rRNA E. coli gene, the mode values obtained for each primer enabled us to allocate them to one of the gene regions defined for that organism by Baker et al. [55]. The reference sequence utilised had 1542 bps distributed in 10 conserved (C1–C10) and nine hypervariable regions (V1–V9). The sequences of these selected primer pairs are described in Additional file 15
ALC amplicon length category, bps base pairs, F forward, KP Klindworth primer, OP oral primer, R reverse, SC coverage at the species level

ISSN: 2049-2618