Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity

The genetic diversity of a vesicular stomatitis virus population was analys ed by RT-PCR, cloning and sequencing of two similar to 500 nucleotide regio ns of the virus genome. PCR amplifications were performed in parallel exper iments with both Tag and Pfu DNA polymerases, and important differences wer e observed. Between 10 and 22 mutations were detected when virus population s were analysed by Tag amplification (20 clones from each region), whereas amplification of the same samples with Pfu revealed between 0 and 5 mutatio ns. PCR fidelity assays, performed under the same PCR conditions as those u sed in the population analysis, showed that the Taq error-rate estimate of 0.27 x 10(-4) misincorporations per bp per cycle was within the range estim ated elsewhere from PCR amplification of recombinant plasmids (0.27-0.85 x 10(-4) errors per bp per cycle) or from functional assays (0.2-2 x 10(-4) e rrors per bp per cycle), The error rate of Taq was found to be 9.3 times hi gher than the error rate of Pfu with DNA as a template, and about 10 times higher with cDNAs obtained by reverse transcription of viral RNA templates from natural populations. In the present study, we discuss (i) the implicat ions of Tag errors on the analysis of genetic variability, based on both th e frequency and nature (replacement vs synonymous) of the observed substitu tions and (ii) the sample size required to assess the genetic variability i n a virus population generated by a single infection.