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

Species-specific probe pairs were designed in a single round, starting from the whole dataset of 352 ClustalWaligned cyanobacteria 16S rRNA sequences, imported, converted and grouped into 18 group-specific consensus sequences by ORMA. Calculated consensus sequences were highly similar, (ClustalW score = 87.31 AE 2.13, n = 18), had a high consensus score (average score 89.20 AE 4.16, n = 352) and a very low content of degenerated bases (average < 2%, max = 6%). ORMA identified a total of 192 candidate probe pairs for the 18 species, with an overall duration of the whole procedure of less than 5 min (Table 2 ). More tests on speed performances of the SBS algorithm on simulated data available as Supplementary Data and Supplementary Figure 2 . One probe pair per species was chosen, according to its ranking after ORMA filtering steps. The probe pair for Anabaena + Aphanizomenon group was flagged as inadequate by ORMA, having six degenerated bases in the CP, which could negatively influence its thermodynamical properties. However, this probe pair insisted on the only discriminating position found for that cluster. The mix containing all probe pairs was tested on the corresponding synthetic templates and, as expected, all except Anabaena+Aphanizomenon gave positive results. Duplicate LDR experiments on 18 probe pairs (17 species-specific + 1 universal) were carried out on 14 16S rRNA PCR products. We performed side-by-side tests of the same DNA samples by the two probe pairs datasets, ORMA and the one described in Castiglioni et al., comparing their performances and specificity. Probe pairs used in Castiglioni et al. identified correctly (P < 0.005, average beta power of the test: 0.85) 6 out of 14 analyzed DNAs (in both duplicate LDR), whereas other two completely failed. Six other DNAs somehow showed a degree of aspecificity (i.e. the correct probe pair was present, but non-specific probe pairs were also called present) (Table 1, Figure 2 ). Cyanobacteria universal probe pair was called as statistically over the background in all the experiments. Evaluations on ratio of signal intensities suggested that hybridizations went well and were not responsible for the aspecificity. In fact, excluding non-specific signals, SNR np had an average value of 1.18 AE 0.61 and SNR p varied between 10 and 680, with an average of about 149 (data not shown). The Anabaena + Aphanizomenon probe pair of Castiglioni et al. study resulted specific on both synthetic and environmental samples (data not shown). This probe pair, however, was designed with its DS insisting on a position which did not discriminate univocally the Anabaena + Aphanizomenon consensus from the consensuses of the other species. Thus, it would never be identified by ORMA as discriminating (because of the way the algorithm is built). Instead, the presence of some internal mismatches (especially the one on the second base before the 3 0 -end of the DS) is probably the reason for this finding. In fact, the mismatch gives instability to the 3 0 -end of the DS when annealing on the 16S rRNA sequences of species other than those of Anabaena + Aphanizomenon cluster, impeding the ligase to join the two adjacent end of the DS and CP oligonucleotides.