SVARAP and aSVARAP: simple tools for quantitative analysis of nucleotide and amino acid variability and primer selection for clinical microbiology

Background: Simple computerized methods that analyse variability along alignments of nucleotide or amino acid sequences can be very useful in a clinical microbiology laboratory for two main purposes. First, to optimize primer selection, which is critical for the identification of infectious pathogens based on gene sequencing: primers must target conserved nucleotide regions bordering highly variable areas to ensure discrimination of species. Second, it can be of interest to reveal mutations associated with drug resistance of pathogen agents. Our aim was therefore to test easy and cost-free tools (SVARAP and aSVARAP) that require short hands-on work, little expertise, and which allow visual interpretation and statistical analysis of results. Results: We first tested SVARAP to improve a strategy of identification of streptococci species of the Viridans Group targeting the groESL gene. Two regions with < 500 nucleotides were identified, one being significantly more discriminant than one of a similar length used in a previous study ( mean number of nucleotide differences between species, 113 ( range: 12-193) vs. 77 ( range: 14-109); p < 10(-3)). Secondly, aSVARAP was tested on reverse transcriptase (RT) sequences from 129 HIV-I clinical strains to identify natural polymorphisms and drug- selected mutations emerging under nucleoside RT inhibitor (NRTI)-selective pressure. It revealed eleven of the 18 RT mutations considered in a reference HIV-I genotypic NRTI-resistance interpretation algorithm. Conclusion: SVARAP and aSVARAP are simple, versatile and helpful tools for analysis of sequence variability, and are currently being used in real practice in our clinical microbiology laboratory.