Simulation of the expected fraction of cells with deviant karyotype as a function of generation time and chromosome mis-segregation rate was performed as follows. A seeding cell was represented as a vector of length 16, each element of which represented the copy number of one of the 16 yeast chromosomes. At each generation, each cell was allowed to self-duplicate and during this process each of the 16 duplicated chromosomes was allowed to mis-segregate with a given probability, resulting in one daughter inheriting both copies and the other daughter not inheriting any copy of that particular chromosome. At the end of each generation, cells that lost all copies of any given chromosome were discarded as dead cells. Every time the simulated colony reached .100,000 cells, a random sampling of 100,000 cells was used to simulate the next generation to limit computational complexity. As a control, the same simulation was performed using different cell number cutoffs without significant differences (data not shown). Simulation of the expected distribution of apparent ploidy of spores from triploid meiosis was performed as follows. As above, random spores were represented as vectors of length 16, in which each element represented one of the 16 yeast chromosomes and having identical and independent probability of being of copy number ‘1’ or ‘2’. The apparent ploidy of each random spore was calculated based on the known length in base pairs of each of the 16 yeast chromosomes. 10,000 independent simulations were performed, in each of which 41 random spores were generated, i.e. the same number as the experimentally determined ones. Karyotype network reconstruction and determination of level of CIN Chromosome copy number data from both the g20 population and from the 11 colonies analyzed at either g20, g25 or g30 were combined into a matrix of size 12616, in which each row represented one of the 12 ‘individuals’ and each column represented one of the 16 ‘loci’ carrying one of two ‘alleles’, corresponding to the two copy number states (i.e. ‘1’ and ‘2’). This matrix was used as input for the Network software (version 4.5.1.6.), which reconstructed the most likely karyotype network by minimizing the number of allelic changes (here: chromosome copy number gain/loss events) across the entire map. Karyotype changes involving more than one chromosome copy number change were scored conservatively as a single event, as we ignored whether the multiple chromosome mis-segregations occurred in a single erroneous mitosis or multiple subsequent mitotic events. Also, karyotype changes unlikely to have originated directly from the inferred original spore karyotype, but more likely to have originated from one of its karyotypically deviant progeny according to the reconstructed karyotype network, were not counted for the determination of the level of CIN of the ancestral karyotype, as they would be more reflective of the level of CIN of one of its karyotypic deviants as opposed to the level of CIN of the ancestral karyotype. Supporting Information Figure S1 Diagram explaining the number of aneuploid strains analyzed at each step of this study. Reasons for discarding specific strains for subsequent analyses are given on the right. (PDF) Figure S2 Computer simulations of fraction of cells with deviant karyotype as a function of chromosome mis-segregation rates. (A– B) Fraction of cells with deviant karyotypes after 20 (A) or 30 (B) generations. Chromosome mis-segregation rates are indicated on the x-axis whereas the percentage of cells with deviant karyotype is Karyotypic Determinants of Chromosome Instability indicated on the y-axis. Box-plots represent median (thick horizontal bar), inter-quartile range (rectangular square) and outliers (circles) of 30 independent simulations. See Materials and Methods for details on the computer simulation. (PDF) Figure S3 DNA content profiles of all 47 g20 populations analyzed by FACS. FACS profiles of the indicated strains from the g20 population samples are shown in separate panels. The profile of a haploid control strain run in parallel is superimposed on each plot. (PDF) Figure S4 Comparison of observed and expected distribution of apparent ploidies from aneuploid spores obtained by triploid meiosis. Apparent ploidy data was derived from the mode of the G1 peak position (measured by FACS analysis) of all 47 analyzed viable spores obtained by meiosis of a homozygous triploid strain in comparison to the mode of the G1 peak position of a control haploid strain run in parallel. Simulated ploidy data was obtained by computer-generated random karyotypes as explained in the Materials and Methods. Empirical cumulative distribution func- tions are shown for both datasets and their statistical difference was tested by means of a Kolmogorov-Smirnov test. (PDF) Figure S5 Karyotypes of the 41 g20 populations. Each panel shows the absolute chromosome copy numbers determined for each of the 41 karyotyped strains, by combining information from high-throughput FACS and qPCR assays. The 27 strains further processed for analysis, the 6 strains discarded because of redundancy (i.e. part of a pair of siblings with identical karyotype) and the 8 strains discarded because of excessive heterogeneity are each labeled accordingly. (PDF) Figure S6 FACS profiles of the g20 population sample and 11 g20 colony samples. FACS profiles are overlaid and the coefficient of variation (CV) is calculated between the G1 peaks of the 12 samples. (A) Wild type haploid G1 peaks and CV; (B) an example of a strain (s203) with sharp G1 peaks and low CV; (C) an example of a strain (s236) with wide G1 peaks and large CV. (PDF) Figure S7 Karyotype information and karyotype networks of all 27 analyzed aneuploid strains. For all 27 analyzed aneuploid strains, karyotype makeups and reconstructed karyotype networks are shown. The number of CIN events used to qualitatively classify the aneuploid strains is shown on the right. See legend of Figure 2 for details on data presentation. Note that there exist two equally probable karyotype networks for strain 252, however in both cases the number of CIN events directly linked back to the original karyotype are the same hence its CIN classification is not affected. Alternative network is indicated by dashed lines. (PDF) Figure S8 Distribution of genes implicated in CIN across the 16 yeast chromosomes. For each of the 16 yeast chromosomes, the y coordinate represents the number of genes belonging to a specific class (identified on the y-axis of the diagram) present on the chromosomes and the x coordinate represents the total number of protein-coding genes on the same chromosome. The dashed line represents the expected number of genes in each class based on the assumption of uniform distribution across the 16 chromosomes. Chromosome V and chromosome VII are highlighted in red and green respectively. (PDF) PLoS Genetics | www.plosgenetics.org 12 May 2012 | Volume 8 | Issue 5 | e1002719