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-1 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-1 genotypic NRTI-resistance interpretation algorithm.


CONCLUSIONS
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.
Colson
Philippe
P
Laboratoire de Virologie, Fédération Hospitalière de Bactériologie-VirologieClinique et d'Hygiène, CHRU Timone, 264 rue Saint-Pierre 13385, Marseille cedex 05, France. philippe.colson@ap-hm.fr
Tamalet
Catherine
C
Raoult
Didier
D
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D018833:Chaperonins / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers; D004352:Drug Resistance, Microbial / Q000235:genetics*; D024882:Drug Resistance, Viral / Q000235:genetics; D014644:Genetic Variation; D054303:HIV Reverse Transcriptase / Q000235:genetics; D008828:Microbiological Techniques; D009154:Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D016415:Sequence Alignment / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods; D012984:Software; D034363:Viridans Streptococci / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D017931:DNA Primers; C097970:GroESL protein, Bacteria; D054303:HIV Reverse Transcriptase; D018833:Chaperonins