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

Searching, designing and selecting oligonucleotide probes for molecular applications experiments on sets of highly similar sequences, such as the 16S rRNA, is a non-trivial procedure, which involves many complex and timeconsuming steps. In this article, this procedure was accomplished by the use of ORMA, an integrated architecture of Matlab scripts. The 16S rRNA, a gene sequence of more than 1500 bp, is the preferred genomic target for analyses in the microbiological field (17) (18) (19) (20) . It should be noted that 16S region is commonly used in taxonomical classifications involving in silico alignment and procedures for its two basic properties: (i) 16S presents highly conserved regions which can be used to correctly align all the sequences in the database; (ii) on the other side, 16S presents highly polymorphic regions that can be used in clusterization, phylogenetic tree construction and molecular discrimination of microbiological families even very close one to each other (32) . Use of an automated method for discriminating positions determination, probe retrieval and filtering has obvious and evident advantages over the manual design, often used in previously published papers (8, (33) (34) (35) . These advantages become more significant with increasing dimension of the databases and of the sequences length. ORMA can perform all these operations with user-specified parameters in an automated way and calculates a series of Where sequencing has been performed, the result of the classification is also reported. Sample ID refers to the numbers used in Figure 2 .