ORMA: a tool for identification of species-specific variations in 16S rRNA gene and oligonucleotides design

Severgnini, Marco; Cremonesi, Paola; Consolandi, Clarissa; Caredda, Giada; De Bellis, Gianluca; Castiglioni, Bianca

Cyanobacteria dataset experiment. The complete cyanobacteria 16S rRNA data set comprised a total of 352 sequences, which were organized by phylogenetical similarity and grouped in a total of 18 clusters, as described in (8) . Multiple alignments of all the sequences was performed by ClustalW (26) and the resulting file was imported into ORMA, where 18 consensus, one per cluster, were built. Consensus sequences were determined following the 'ARB-like' algorithm (as described in 'Materials and Methods' section and in Supplementary Methods), setting 50% as the threshold for gaps and 40% as the threshold for other bases. Melting temperature calculations followed the 'salt-adjusted' method, with 50 mM Na + and 0% formamide. Candidate probe pairs were filtered on the basis of their length (minimum 25 nt, maximum 60 nt per probe), melting temperature (63-688C) and number of degenerated bases (maximum 4), on both DS and CP. The best probe pairs for all the species were selected, according to their best intra-and inter-group scores. We required that no less than 80% of the sequences constituting each of the 18 clusters carried the same base as the consensus in the candidate discriminating position (intra-group score). When only one candidate was designed or the intra-group score of the best candidate was below 80%, we still picked that candidate for further evaluations. On the other hand, the inter-group score was set to be below a 2% threshold, with the same exceptions as above. The 'Unicyano' probe, which allowed the identification of any of the species in the study, was the one proposed by Castiglioni et al., with minor refinements for adjusting its melting temperature. At first, the LDR mix made by all probe pairs (250 fmol/ml each probe) was tested on specific synthetic templates (perfectly complementary to each probe pair) to assess the feasibility of the LDR procedure with the ORMAdesigned probe pairs. Then, a total of 14 DNA samples, corresponding to 13 cyanobacteria species (kindly provided by MIDI_CHIP project partners, http://www.cip .ulg.ac.be/midichip/) (Table 1) , were tested in duplicate, independent, LDR experiments, with both ORMA and Castiglioni et al. probe pairs.