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

In this case, the discrimination power resides more in the decreased melting temperature of mismatched duplexes, rather than on a perfectly matched base pair between the probe and the target. Although our tool was applied on the design of probes for a specific technique (LDR) and on a specific target gene (16S rRNA), the software is not limited to this combination. LDR technique approach implied the retrieval of a pair of sequences, one of which (the DS probe) insisted on the discriminating position, whereas the other (the CP probe) is designed to anneal one base 3 0 -downstream of the discriminating position; the design of probes for minisequencing application would have implied only the determination of one probe with its 3 0 -end one base before the variation. At the same time, the design of a reporting probe for a TaqMan Real-time PCR assay would have implied the determination of one oligo with the single-base variation in the middle of the sequence. Due to its modular structure and to the straightforwardness of other applications from the already implemented one, probe sets retrieval and filtering methods could be easily added, starting from the discriminating positions found by the SBS algorithm. A further extension to hybridization probes/standard PCR primer design will be evaluated, changing the strategy for determining the positions to be tested. Provided that the initial database of sequences is accurate, updated and complete as much as possible, ORMA can retrieve discriminating positions and design specific probes on every set of sequences. Its implementation, in fact, is not based on an internal database of sequences (which are, instead, retrieved and loaded from external resources) and can be extended to any gene. In any case, the database should be critically built by only context-specific sequences. Standard procedures, like PCR with specific primers, can help in isolating only the subsets of sequences which constitute the actual database from those completely unrelated to the biological context under investigation, avoiding any interference with actual probes, as exemplified by the cyanobacteria dataset experiment. Sequences of off-target or distantlyrelated species could negatively act in the process of multiple-alignment, leading to poorly aligned datasets and biased designs. Since the databases, cyanobacterias in particular, are constantly and frequently upgraded, ORMA capability of determining discriminating positions can be refined, depending on the completeness of the initial datasets (both positive and negative set). Moreover, the continuous changing of classification and the addition of new sequences make an exhaustive and definitive design of the best cyanobacteria probes absolutely not trivial.