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

(iv) Discriminating position related filters and scores. The putative discriminating positions and related candidate probes are subjected to a series of constraints and quality filters. The software keeps track of all the designed candidates, assigning a quality score, depending on how many filters they pass. The current options of the script on the discriminant base are: (a) limiting the range of positions, in order to exclude candidates insisting on positions too close to the 5 0 -or 3 0 -end of the sequences, where, usually, the majority of errors in the alignment or characterization of the sequences occur and (b) testing the presence of other species with probes insisting on the same position, thus excluding eventual interactions between a single CP and multiple DS, with subsequent non-specificity. The candidate probes can also be filtered and ranked according to their thermodynamic properties (length, melting temperature, number of degenerated bases, low complexity regions), evidencing the candidates having a certain length, a melting temperature comprised in a user-specified range, having no more than the inputted number of degenerated bases (which can be a real issue for the oligonucleotide specificity), having short homopolymeric regions and not comprising short tandem repeats. Then, ORMA calculates some specific statistics for the qualitative evaluation of the candidates designed on consensus sequences, compared to the original dataset (i.e. the subset of sequences from which every consensus is built): (a) the intra-group score, as the number of initial sequences having the same discriminating base as the consensus and (b) the inter-group score, as the number of sequences other than those used for that consensus having the same discriminating base as the candidate one. This latter score is calculated only when the consensus were created inside ORMA, starting from a single-global alignment. These scores allow the choice of probes that best discriminate between the target and the non-target sequences (i.e. having the highest intra-group and the lowest inter-group score). The software output can be exported as a comma-separated spreadsheet reporting: (a) the list of all the discriminating bases, grouped per species, with absolute (referring to the global alignment) and relative (referring to the specific consensus) positions of the discriminating base, and the base distributions of all the other consensus sequences in the same position; (b) the thermodynamic parameters of the candidate probe pairs, including the T m , the length of DS and CP probes and the number of degenerated bases in each and (c) the qualitative filtering and the specificity-related scores, including the sequence score, as the average of the consensus scores along all the bases constituting the DS and CP, with penalties for degenerated bases.