Heterozygous BMN lines

Optical density at 80 hours 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 0.8 0.6 0.4 0.2 a YPD b YPD + 1µM nystatin c YPD + 2µM nystatin d YPD + 4µM nystatin 1 3 5 6 9 13 16 20 21 22 23 25 28 29 30 31 32 33 34 35 Heterozygous BMN lines Supplementary Figure 1 Stochastic growth of heterozygous BMN lines in the second deep well box experiment. All heterozygous BMN lines were grown in the presence of a) YPD, b) YPD + 1µM nystatin, c) YPD + 2µM nystatin, and d) YPD + 4µM nystatin for 80 hours. Culture from wells that showed growth in this experiment were frozen and utilized for Sanger sequencing. 1

log(optical density) a b Initial growth in YPD+2µM nystatin homozgyote heterozygote * Isolated after initial growth, culture from * 0 15 30 45 60 75 Time (hours) Supplementary Figure 2 Elimination of stochastic growth after isolation of het-grow lines. Culture isolated from heterozygous replicates (het-grow lines) that a) showed stochastic growth in YPD + 2µM nystatin, b) grew much less stochastically after they were isolated, propagated for 24 hours of growth in YPD, then inoculated back into nystatin. 2

a BY4741: ERG5/ERG5 b BMN35 heterozgote: ERG5/erg5Δ deletion... c Polymorphic population: ERG5/erg5Δ & erg5δ/erg5δ deletion... Supplementary Figure 3 Chromatograms indicate that BMN35 replicate populations isolated after growth in YPD + 1µM nystatin are polymorphic. A comparison of chromatograms from a) BY4741 (wildtype) culture, b) heterozygous BMN35 culture and c) culture isolated after BMN35 heterozygotes were grown in YPD + 1µM nystatin indicates that these populations are polymorphic for homozygous and heterozygous individuals carrying the deletion that is present in the BMN35 background. The peak heights that correspond to the wildtype base in the appropriate region are less than 50%, indicating that homozygous mutant individuals are sweeping through the population. 3

BMN line colony 3 188 9 12 9 130 13 54 23 144 28 14 Coverage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Chromosome Supplementary Figure 4 Average depth of coverage for each chromosome in six het-grow lines. Six het-grow lines (those that exhibited the lowest G1 mean) were Illumina resequenced and coverage per chromosome was obtained using configurebuild.pl from Illumina s CASAVA-1.8.0 package. Plotted for each strain is the proportion of sequenced sites that map to each chromosome relative to the proportion of known mapped sites on that chromosome within the reference genome. Line is diploid according to flow cytometry yet monosomic for chromosome 3, while line is haploid by flow cytometery, but disomic for chromosomes 2 and 9. 4

2.5 A-I A-V URA3 A-IX A-XIII ERG5 ERG6 0 0.10 0.20 2.5 A-II SCO1 0.2 0.4 A-VI 0.1 0.2 0.3 0.4 A-X 0.2 0.4 0.6 0.8 A-XIV Depth 2.5 0.2 0.4 0.6 0.8 A-III LEU2 MAT 0 0.10 0.20 A-VII 0.2 0.4 0.6 A-XI 0.2 0.4 0.6 0.8 A-XV HIS3 0 0.10 0.20 0.30 2.5 A-IV 0.4 0.8 A-VIII ERG7 CUP1 0.2 0.4 0.6 A-XII ERG3 RDN1 repeat MET15 0.4 0.8 A-XVI 0.2 0.4 Chromosome 0.4 0.8 0.2 0.4 0.6 0.8 5

2.5 B-I B-V URA3 B-IX B-XIII ERG5 ERG6 0 0.10 0.20 2.5 B-II SCO1 0.2 0.4 B-VI 0.1 0.2 0.3 0.4 B-X 0.2 0.4 0.6 0.8 B-XIV Depth 2.5 0.2 0.4 0.6 0.8 B-III LEU2 MAT 0 0.10 0.20 B-VII 0.2 0.4 0.6 B-XI 0.2 0.4 0.6 0.8 B-XV HIS3 2.5 0 0.10 0.20 0.30 B-IV 0.4 0.8 B-VIII ERG7 CUP1 0.2 0.4 0.6 B-XII ERG3 RDN1 repeat MET15 0.4 0.8 B-XVI 0.2 0.4 Chromosome 0.4 0.8 0.2 0.4 0.6 0.8 Supplementary Figure 5 Depth of coverage across the genome for six het-grow lines. For each chromosome in each line, alignments were obtained using Illumina s CASAVA-1.8.0 package. The pileup program in samtools-0.1.7a was then used to obtain reads by position across the genome. A custom perl script was then used to calculate average depth of coverage in 1000 basepair windows across each chromosome. These windows skipped over sites where the depth of coverage was zero, which could be due to either deletions or ambiguous alignments (plots showing positions with no alignments are available upon request). Panels A-i through A-XVI illustrate the depth of coverage for chromosomes I-XVI for lines,, and. Panels B-i through B-XVI illustrate depth of coverage for lines (note extra copies of chromosomes 2 and 9),, and (note missing copy of chromosome 3). 6

a Ergosterol genes ERG7 ERG6 ERG3 ERG5 Coverage b Control genes FRA1 DIC1 RCE1 DAT1 BMN13 54 BMN23 144 BMN3 188 BMN19 130 BMN28 14 BMN9 12 Supplementary Figure 6 Gene copy coverage for ergosterol and control genes from in silico qpcr. Fragment counts from the beginning and end of each gene obtained by in silico qpcr. The unix command grep was used to count the number of copies of the first and last 18bp of each ERG gene, in forward and reverse orientations, directly within the fastq sequences of unaligned 100bp paired end fragments obtained from Illumina sequencing. The average number of fragment counts was then divided by the average coverage across the genome from the CASAVA alignments. This relative fragment number is plotted, with 1 on the y-axis representing the median across the 6 lines (to minimize differences in scale across the genes). Panels show the relative fragment number from in silico qpcr of the (top) ERG genes and (bottom) four control genes chosen from the same chromosomes as ERG3 and ERG6. For each gene, we confirmed by BLASTing the yeast genome (http://www.yeastgenome.org) that the 18bp at the beginning and end of each gene was unique. 7

Supplementary Table 1 Statistical comparison of maximal growth rates among wildtype (aa), heteorzygous (Aa) and homozygous mutants (AA) grown in a permissive environment (YPD). Tukey Test result BMN line Gene ANOVA Aa vs. AA Aa vs. wt AA vs. wt 1 ERG7 F 2,12 = 12.45, p = 012 p = 009 p = 0.20 p = 22 3 ERG6 F 2,12 = 12.24, p = 013 p = 0.15 p = 32 p = 01 5 ERG6 F 2,12 = 9.27, p = 037 p = 044 p = 0.77 p = 15 6 ERG6 F 2,12 = 19.95, p < 001 p = 0.43 p = 005 p < 001 9 ERG6 F 2,12 = 9.96, p = 028 p = 036 p = 0.80 p = 11 13 ERG6 F 2,12 = 0.43, p = 025 p = 033 p = 0.82 p = 095 16 ERG6 F 2,12 = 28, p = 001 p = 001 p = 0.13 p = 038 20 ERG6 F 2,12 = 16.30, p = 004 p = 004 p = 1 p = 028 21 ERG3 F 2,12 = 28.51, p < 001 p < 001 p = 23 p = 022 22 ERG3 F 2,12 = 19.93, p = 002 p = 0.34 p = 017 p = 002 23 ERG3 F 2,12 = 19.95, p = 002 p = 0.22 p = 024 p = 001 25 ERG3 F 2,12 = 12.35, p = 012 p = 011 p = 0.39 p = 122 28 ERG3 F 2,12 = 38.66, p < 001 p < 001 p = 14 p = 005 29 ERG3 F 2,12 = 15.75, p = 004 p = 003 p = 92 p = 17 30 ERG3 F 2,12 = 22, p = 002 p = 002 p = 0.35 p = 016 31 ERG3 F 2,12 = 22.51, p < 001 p < 001 p = 75 p = 033 32 ERG3 F 2,12 = 17.33, p = 003 p = 22 p = 43 p = 002 33 ERG3 F 2,12 = 24.07, p < 001 p < 001 p = 29 p = 052 34 ERG3 F 2,12 = 14.63, p = 007 p = 0.89 p = 022 p = 01 35 ERG5 F 2,12 = 0.43, p =0.66 p = 0.64 p = 0.94 p = 0.83 8

Supplementary Table 2 Statistical comparison of biomass production (optical density at 24 hours) among wildtype, heterozygous and homozygous mutants grown in an unstressful environment (YPD). Tukey test result BMN line Gene ANOVA Aa vs. AA Aa vs. wt AA vs. wt 1 ERG7 F 2,12 = 7.82, p = 067 p = 0.35 p = 7 p = 05 3 ERG6 F 2,12 = 28.73, p < 001 p = 004 p = 0.22 p < 001 5 ERG6 F 2,12 = 7.71, p = 070 p = 0.11 p = 0.24 p = 054 6 ERG6 F 2,12 = 23.05, p < 001 p = 20 p = 093 p < 001 9 ERG6 F 2,12 = 6.04, p = 15 p = 0.12 p = 0.44 p = 13 13 ERG6 F 2,12 = 82.44, p < 001 p < 001 p = 094 p < 001 16 ERG6 F 2,12 = 40.30, p < 001 p < 001 p 0.82 p < 001 20 ERG6 F 2,12 = 13.07, p = 01 p = 07 p = 3 p = 010 21 ERG3 F 2,12 = 109.67, p < 001 p < 001 p = 12 p < 001 22 ERG3 F 2,12 = 206.51, p < 001 p < 001 p = 003 p < 001 23 ERG3 F 2,12 = 182.87, p < 001 p < 001 p = 010 p < 001 25 ERG3 F 2,12 = 157.13, p < 001 p < 001 p = 22 p < 001 28 ERG3 F 2,12 = 103.71, p < 001 p < 001 p = 0.30 p < 001 29 ERG3 F 2,12 = 87.15, p < 001 p < 001 p = 0.20 p < 001 30 ERG3 F 2,12 = 129.34, p < 001 p < 001 p = 01 p < 001 31 ERG3 F 2,12 = 167.69, p < 001 p < 001 p = 58 p < 001 32 ERG3 F 2,12 = 124.93, p < 001 p < 001 p = 0.22 p < 001 33 ERG3 F 2,12 = 165.58, p < 001 p < 001 p = 0.77 p < 001 34 ERG3 F 2,12 = 13.32, p = 009 p = 3 p = 065 p = 01 35 ERG5 F 2,12 = 2.75, p = 0.10 p = 0.88 p = 0.23 p = 0.11 9

Supplementary Table 3 Primers used to re-sequence the original mutations in the het-grow lines. Gene BMN lines Primers Amplicon size ERG7 BMN1 F 5 -CCTGCTGAAGTTTTTGGTAACAT-3 826 R 5 -AAGCCGTGCTAAAGGATAAATGG-3 ERG6 BMN3, 5, 6, 9, 13, 20 F 5 -TTCGGGTGTTTTCTCCTATCC-3 1455 R 5 -GTGGGGTAGTAAAGGCATCG-3 ERG3 BMN21, 22, 23, 28, 30, 31, 32 F 5 -GGTTGCAGAGGAGGTCAGTT-3 1241 R 5 -CGTTATTCGTTGTGTGAAAGTT-3 ERG5 BMN35 F 5 -GCAAGCAGGGAATCTTGTTG-3 1857 R 5 -TCAAAACGCCAACCCTTAAT-3 10

Supplementary Table 4 Het-grow colonies isolated and Sanger sequenced after growth in YPD in bioscreen wells or deep well boxes. All lines were initially heterozygous for a particular ergosterol mutation; the third column reports their genotype after 24 hours. Colony numbers refer to samples isolated and frozen from the second bioscreen (b) and second deep well box (d) experiments. Genotype at Growth BMN line Gene initial locus experiment Colony 1 ERG7 het d 7A 3 ERG6 het d 9B 6 ERG6 het d 8C 9 ERG6 het d 5C 13 ERG6 het d 10A 20 ERG6 het d 2C 21 ERG3 het b 13 21 ERG3 het b 93 23 ERG3 het b 14 23 ERG3 het b 94 32 ERG3 het b 17 32 ERG3 het b 97 35 ERG5 het d 10B 11

Supplementary Table 5 Het-grow colonies isolated and Sanger sequenced after growth in YPD+1µM nystatin in deep well boxes. All lines were initially heterozygous for a particular ergosterol mutation; the third column reports their genotype after 80 hours. Colony numbers refer to samples isolated and frozen from the second deep well box experiment. Genotype at Growth BMN line Gene initial locus experiment Colony 1 ERG7 hom d 7E 1 ERG7 hom d 7G 3 ERG6 hom d 9B 5 ERG6 hom d 3A 5 ERG6 hom d 3C 9 ERG6 hom d 5A 9 ERG6 hom d 5C 13 ERG6 hom d 10A 13 ERG6 hom d 10C 13 ERG6 hom d 10G 20 ERG6 hom d 2A 20 ERG6 hom d 2C 21 ERG3 hom d 4F 21 ERG3 hom d 4H 22 ERG3 hom d 6H 23 ERG3 hom d 5B 23 ERG3 hom d 5D 28 ERG3 hom d 6A 28 ERG3 hom d 6C 30 ERG3 hom d 11A 30 ERG3 hom d 11C 31 ERG3 hom d 2B 31 ERG3 hom d 2D 32 ERG3 hom d 8B 32 ERG3 hom d 8D 35 ERG5 het d 10B 35 ERG5 het d 10D 35 ERG5 het d 10F 35 ERG5 het d 10H Based on the height of the chromatogram trace obtained by Sanger sequencing, the mutant allele frequency in these populations is greater than, an indication that homozygous mutant individuals are present. 12

Supplementary Table 6 Het-grow colonies isolated and Sanger sequenced after growth in YPD+2µM nystatin in bioscreen wells or deep well boxes All lines were initially heterozygous for a particular ergosterol mutation; the third column reports their genotype after 72 hours of growth in the plate reader (b) or 80 hours of growth in a deep well box (d). Genotype at Growth BMN Gene initial locus experiment Colony 1 ERG7 hom b 52 1 ERG7 hom b 134 3 ERG6 wt b 116 3 ERG6 hom b 188 5 ERG6 hom b 80 9 ERG6 hom b 12 9 ERG6 hom b 130 13 ERG6 hom d 10A 13 ERG6 hom d 10E 13 ERG6 hom b 54 13 ERG6 hom b 136 21 ERG3 hom d 4F 21 ERG3 hom d 4H 21 ERG3 hom b 60 21 ERG3 hom b 106 23 ERG3 wt b 26 23 ERG3 hom b 144 28 ERG3 hom b 14 28 ERG3 wt b 50 32 ERG3 hom b 68 32 ERG3 wt b 186 35 ERG5 wt b 190 13

Supplementary Table 7 Het-grow colonies isolated and Sanger sequenced after growth in YPD+4µM nystatin in bioscreen wells or deep well boxes. All lines were initially heterozygous for a particular ergosterol mutation; the third column reports their genotype after 72 hours of growth in the plate reader (b) or 80 hours of growth in a deep well box (d). Genotype at Growth BMN Gene initial locus experiment Colony 1 ERG7 wt b 16 1 ERG7 hom b 52 1 ERG7 hom d 7A 1 ERG7 hom d 7E 3 ERG6 hom d 9H 3 ERG6 hom d 9F 5 ERG6 hom d 3C 9 ERG6 hom b 84 9 ERG6 hom d 5A 9 ERG6 hom d 5C 13 ERG6 hom d 10E 21 ERG3 hom d 4F 21 ERG3 wt d 4H 22 ERG3 wt d 6B 22 ERG3 hom d 6H 25 ERG3 hom b 30 28 ERG3 hom d 6A 28 ERG3 hom d 6C 31 ERG3 hom d 2B 31 ERG3 hom d 2D 14

Supplementary Table 8 Eight het-grow lines were the result of contamination. Eight het-grow lines were wildtype homozygous for the expected mutation but carried secondary homozygous mutations in either ERG6 or ERG3 that are present in other BMN lines. Expected mutation Observed ergosterol mutation Gene Gene Amino acid Line Colony Environment (location in gene) (location in gene) change BMN1 16 (b) 4µM nystatin ERG7 (bp2096) ERG6 (bp669) Tyr223Stop (same as BMN19) BMN3 116 (b) 2µM nystatin ERG6 (bp131) ERG3 (bp898) Gly300Arg (same as BMN32) BMN21 4H (d) 4µM nystatin ERG3 (bp187) ERG6 (bp220) Tyr74Stop (same as BMN5) BMN22 6B (d) 4µM nystatin ERG3 (bp227) ERG6 (bp642) Leu214Phe (same as BMN16) BMN23 26 (b) 2µM nystatin ERG3 (bp284) ERG3 (bp187) Arg63Stop (same as BMN21) BMN28 50 (b) 2µM nystatin ERG3 (bp640) ERG6 (bp279) Gly127Arg (same as BMN9) BMN32 186 (b) 2µM nystatin ERG3 (bp898) ERG3 (bp615) Trp205Stop same as BMN31) BMN35 190 (b) 2µM nystatin ERG5 (bp252) ERG6 (bp131) Gln44Stop (same as BMN3) 15

Supplementary Table 9 Secondary mutations remained heterozygous. Seven of the original BMN lines contained secondary mutations that were unlinked to the ergosterol mutations. These secondary mutations remained heterozygous in the 24 related het-grow lines isolated from both bioscreen (b) and deep well box (d) experiments. This suggests that LOH was localized to the chromosome and did not arise due to ploidy cycles. Ergosterol gene, Environment Secondary Genome position Experiment BMN line Colony genome position (µm nystatin) gene (Chr.Bp) Mutation type 1 7E ERG7, VIII.241194 1 intergenic XV.193885 C>T d 1 7G ERG7, VIII.241194 1 intergenic XV.193885 C>T d 1 52 ERG7, VIII.241194 2 YOLO73C XV.193885 C>T b 1 134 ERG7, VIII.241194 2 YOLO73C XV.193885 C>T b 1 52 ERG7, VIII.241194 4 YOLO73C XV.193885 C>T b 1 7A ERG7, VIII.241194 4 intergenic XV.193885 C>T d 1 7E ERG7, VIII.241194 4 intergenic XV.193885 C>T d 3 9B ERG6, XIII.252772 1 intergenic XVI.479630 G>A d 3 9H ERG6,XIII.252772 4 intergenic XVI.479630 G>A d 3 9F ERG6,XIII.252772 4 intergenic XVI.479630 G>A d 5 3A ERG6, XIII.252772 1 CDC23 VIII.438829 A>C d 5 3C ERG6, XIII.252772 1 CDC23 VIII.438829 A>C d 5 80 ERG6, XIII.252772 2 CDC23 VIII.438829 A>C b 5 C3 ERG6, XIII.252772 4 CDC23 VIII.438829 A>C d 13 A10 ERG6, XIII.252596 1 intergenic V.74569 C>T d 13 E10 ERG6, XIII.252596 1 intergenic V.74569 C>T d 13 54 ERG6, XIII.252596 2 GDA1 V.74569 C>T b 13 136 ERG6, XIII.252596 2 GDA1 V.74569 C>T b 22 11A ERG3, XII.254087 1 intergenic XV.188974 T>G d 22 11C ERG3, XII.254087 1 intergenic XV.188974 T>G d 23 5B ERG3, XII.254144 1 SCW11 VII.442319 A>G d 23 5D ERG3, XII.254144 1 SCW11 VII.442319 A>G d 23 144 ERG3, XII.254144 2 SCW11 VII.442319 A>G b 25 30 ERG3, XII.254475 4 FCY2 V.267874 G>A b 16