Microfluidics-based automated genotyping of Saccharomyces Cerevisiae strain by interdelta sequence typing : an interlaboratory comparasion

Abstract

High-throughput molecular characterization of microbial isolates requires the application of automated microfluidic electrophoresis. We herein evaluate the factors that affect interlaboratory reproducibility of interdelta sequence typing for Saccharomyces cerevisiae strain delimitation, using microfluidic electrophoresis (Caliper Lab Chip ® ). This approach is necessary for the constitution of bio-databanks, equitable sharing of genotypic data among laboratories, for biodiversity conservation and sustainable development of genetic resources. Delta sequences are 300 bp regions flanking retrotransposons Ty1 and Ty2 of S. cerevisiae, occurring also as separate elements dispersed throughout the genome. PCRbased interdelta sequence typing has a high discriminatory power [1], generating polymorphic banding patterns. Our approach included 12 genetically diverse S. cerevisiae strains, two different Taq polymerases (commercial and in-house cloned/prepared) and two different thermal cyclers. PCR amplifications were performed in two laboratories, resulting in a total of 384 electrophoretic banding patterns (32 replicates for each strain). From the combinations between strains, Taq polymerase, thermal cycler and laboratory, a total of 60 different groups was obtained. Data were analyzed in terms of the fragment sizes (bp), absolute and relative concentrations of each band. Due to the lack of normality (Kolmogorov-Smirnov and Shapiro-Wilk tests) and the homogeneity of variances (Levene's test), the ANOVA test was not applied. The nonparametric alternative, Kruskal-Wallis one-way analysis of variance was used to test the equality of the medians among the different groups. By rejecting the null hypotheses with a p-value < 0.001, we performed multiple pairwise comparisons using the method proposed by Conover and Iman [2], based on a t-Student distribution to search for the origins of the differences. The data obtained revealed that both the performance of experiments in two independent laboratories and the use of different Taq polymerases introduced significant variability between the respective replicates. The use of in-house cloned/prepared Taq polymerase was associated with highest variability, pointing to the need for careful experimental standardization of PCRbased interdelta sequence analysis.