Comparison of two approaches for identification of haplotypes and point mutations in Candida albicans and Saccharomyces cerevisiae.

The human fungal pathogen Candida albicans displays a very high degree of plasticity, including the types of genomic changes frequently observed with cancer cells, such as gross chromosomal rearrangements, aneuploidy, and loss of heterozygosity. Despite its relevance to every aspect of genetics and evolution of this pathogen, our understanding of the mutation process and its bearing on organismal fitness remains quite limited. Here, we have evaluated and compared two approaches to estimate the mutation frequency at three ORFs/regions (HIS4, CEN4 and EST2) of the C. albicans genome. Sequencing of individual DNA molecules (clone-by-clone sequencing) identified de novo mutations at these DNA regions, whose frequency was similar to that observed for S. cerevisiae at homolog sites following the same approach. However, mutations were not detected when the same regions were directly sequenced from the pooled DNA. In addition, in the absence of the homologous recombination protein Rad52, mutation frequency within these sites remained unaltered. The use of an alternative polymerase also found mutations. These results suggest that at least some mutations are artifacts caused by the polymerase used, advising that post-PCR procedures might generate mutations which may become undistinguishable from the genuine mutations and thus may interfere with mutational analysis. Furthermore, we recommend that new mutations found in the sequences of cloned alleles used for the determination of haplotypes should be contrasted with the sequence yielded by the pooled DNA.