Computational protocol: Saccharomyces cerevisiae: Population Divergence and Resistance to Oxidative Stress in Clinical, Domesticated and Wild Isolates

Similar protocols

Protocol publication

[…] Genomic DNAs were diluted 1∶100 and 2 µl added to a 25 µl reaction from TaKaRa Ex Taq kit with a final primer concentration of 0.6 µM (). The PCR regime consisted of 5 min initial denaturation at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at the appropriate annealing temperature (), and 45 sec at 72°C, concluding with 10 min extension at 72°C. PCR products were purified using the Montáge-SEQ96Cleanup Kit (Millipore) or the ExoSAP protocol and their concentration determined electrophoretically . Between 500 and 1000 ng PCR product were sequenced with BigDye chemistry version 3 according to the instructions supplied by Applied Biosystems. Chromatograms were assembled and analyzed in Sequencher 4.8 (Gene Codes Corp.) and sequences edited in MacClade 4.06 . ITS sequence data were blasted against GenBank using the nr database and the blastn algorithm .The haplotype phase of nuclear loci in strains with more than one heterozygous site per locus was determined using PHASE 2.1.1. , . Loci that received low confidence probabilities were cloned using the pCR2.1 TOPO TA Cloning Kit (Invitrogen) and sequenced as described above. Two haplotypes per strain were further analyzed. PCA, HWE, AMOVA, Fst, π, IA were calculated in GenAlEx 6, DnaSP 4.50.3, and MultiLocus 1.3 –. 98.9% confidence envelopes were calculated for three PCA groups based on three times the standard deviation of the PCA scores of strains included in each cluster . Strains with missing data points were excluded from these analyses and statistical significance values were calculated in 999 permutations (Fst), and 10,000 (IA). Due to limited sampling size S. boulardii and beetle gut isolates were not included in HWE, Fst, π and IA calculations. An UPGMA tree based on a pair-wise genetic distances between isolates included in PCA was build in PHYLIP 3.68 . Haplotype networks were inferred from MLS1, ACT1, ADP1, PHD1 and RPB1 in PAUP* 4.0b10 applying the parsimony criterion, conducting heuristic searches, and using tree-bisection-reconnection (TBR) as branch swapping algorithm. These five loci were chosen for analysis based on their prior employment in phylogenetic studies of the Saccharomycetales, population genetic analysis of S. cerevisiae and C. albicans and their differential expression patterns upon phagocytosis by macrophages (ACT1, RPB1 , ADP1 , MLS1 , PHD1 ). […]

Pipeline specifications

Software tools Sequencher, MacClade, BLASTN, GenAlEx, DnaSP, PHYLIP
Applications Phylogenetics, Population genetic analysis
Organisms Saccharomyces cerevisiae, Homo sapiens