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

Standard multiple-alignment formats (Clustal-like, Multi Sequence Files, or aligned FASTA format) can be used. A careful check of multiple alignment scores should be made, in order to avoid designs on sequence datasets of distantly related species, which can occur in base misalignments. The scripts also include a procedure for consensus determination from a set of user-defined sequences, according to four different rules: (a) majority rule, in which the consensus base is the most frequently present in the aligned sequences and no degenerated bases are used. In case of equal occurrences, 'N's are used in the consensus; (b) threshold rule 'simple', which assigns a specific base to the corresponding position in the consensus only if its frequency is above a given threshold. Different thresholds can be set for gaps and bases. Degenerated bases are not used and are substituted by 'N's in the consensus; (c) threshold rule 'complex', which comprises also degenerated bases. The algorithm is the same as point (b) option, but requires a threshold for substituting positions with multiple bases above the threshold with the corresponding IUPAC code degenerated base and (d) ARB-like algorithm, with separate thresholds for gaps and bases. All the bases above the given threshold are used to compute eventual degenerated bases.