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

ORMA designed probe pairs have been capable of correctly identifying (P < 0.005, average beta power of the test: 0.85) 12 out of 14 analyzed cyanobacteria samples, on both replicates. Also in this experimental set, the cyanobacteria universal probe pair was called as statistically over the background in all the experiments (as expected, since this probe pair and the ones used in Castiglioni et al. coincided). Performances of the LDR procedure, in terms of signal-to-noise ratios were comparable to those obtained with the Castiglioni et al. probe set, having a SNR np of 1.1 AE 0.26 and a SNR p ranging from 7 to 387 (average $131) (data not shown), indicating a certain variability. In this case, we had no signs of aspecificity in the experiments (Figure 2 ), even in those cases which were critical with Castiglioni et al. probe pairs. In fact, probes were chosen in order to maximize the intra-group similarity (i.e. having the maximum number of sequences in the positive set carrying the discriminating base) and minimize the possibility of an inter-group cross-talk (i.e. having a minimum number of sequences in the negative set carrying the discriminating base) (Figure 3 ). The average of intra-group scores of the candidates was 95.1% AE 10.1% (n = 17), varying in the range 60-100%. The minimum value was that of the cluster of Gleotheceae, in which we had only five sequences, whereas 13 out of the 17 clusters were characterized by a score of 100%. Intergroup scores, on the other hand, were always very low, with an average of 0.4% AE 1% (n = 17). Thus, where ORMA probe pairs failed, we had a false negative call (with the cyanobacteria universal probe pair called as 'present'), but not a false positive. Experiments on the two Nostoc DNAs gave no results on the species-specific probe pair; anyway the presence of a cyanobacterial DNA was correctly assessed by the Universal probe pair. Sequencing of the two products revealed that one of them has been correctly classified by microbiological methods, whereas the other DNA was very uncertain and classified as 'cylindrospermum' (58% confidence) by RDP 'Classifier' tool, release 9.60. [On 22 May 2008, RDP II database for cyanobacteria (release 9.61) underwent a major change in hierarchical classification of the species. The taxonomies here presented refer to older versions, which at present, can be found within genus GpI of On the x-axis, the IDs of the tested samples (see Table 1 for full description) are reported. On the y-axis, the probe pair name is reported. The line 'Other' represents the mean of all the remaining Zip-codes in the universal arrays that were not associated to any actual probe. Experiments on Nostoc samples were repeated twice on different DNAs because of the failure of the first test. Halotolerans probe pair in one replicate of sample 8 (classified as Nostoc) has a P-value of 0.02, above the threshold of 0.01 chosen for significance.