Supplementary Material S2: Evidence for aneuploidy
For: Gerstein, A. C., R. M. McBride, and S. P. Otto (2007) Ploidy Reduction in
Saccharomyces cerevisiae.
5
Comparative genomic hybridization (CGH) was performed as follows (Hughes et
4N
3N
3N
3N
3N
al. 2000) on the five founder colonies used in this experiment ( P300
, R300
, S 300
, R500
, R300
),
on four t = 186 colonies where ploidy reduction was observed, and on the haploid and
tetraploid ancestral colonies from Gerstein et al. (2006).
each case, the control colony
 In 
  
was a distantly related haploid strain (YPH499). Single colonies were isolated from
10
frozen stock and grown to stationary phase in liquid YPD. Genomic DNA from 8 ml of
stationary phase culture was extracted using a standard yeast mini-prep DNA isolation
22.5% of 20 kHz using a Micro Ultrasonic Cell Disrupter (Kontess) to obtain DNA
fragments of ~0.1–10 kilobases and purified with a QIAquick PCR Purification Kit
15
(Qiagen). The haploid control and experimental DNA samples were labeled with Cy3 or
Cy5 using the Mirus Label IT Nucleic Acid Labeling Kit (Mirus), according to the
manufacturer’s protocol. Each CGH experiment was repeated with dye-swapping. Cylabeled samples were purified using a CyScribe GFX Purification kit (GE Healthcare).
The labeled genomic DNA was co-hybridized to S. cerevisiae microarrays obtained from
20
the University Health Network Microarray Centre (Toronto). The slides were washed and
scanned using ScanArray Express (PerkinElmer). ImaGene (BioDiscovery, Inc.) was
used to quantify the relative fluorescence of Cy3 and Cy5.
In the analyses of the CGH experiments, chromosomes were identified as
aneuploid if their fluorescence ratio departed by more than 10% from the expected ratio
25
of one and if their fluorescence ratio lay outside of a rejection region. Only
chromosomes exhibiting a substantial departure from the expected fluorescence ratio of
one (>10%) were considered as potentially aneuploid for two reasons. First, slight
departures from a ratio of one could be caused by small-scale genetic divergence (indels,
nucleotide substitutions) between the experimental and control lines. Second, spatial
30
variation in hybridization signal across a microarray could cause departures from the
1
expected ratio of one, because the oligonucleotide sequences were arrayed non-randomly
on the microarrays obtained from the University Health Network Microarray Centre
(Toronto), with clustering of sequences from the same chromosomes (see Gerstein et al.
2006).
5
In addition, a rejection region was constructed to identify chromosomes
exhibiting statistically significant evidence of aneuploidy by adding  t 0.0016,15 SE pooled
to the expected log-fluorescence of zero. Because a standard error ( SE ) based on two
microarrays is subject to substantial error, a pooled standard error ( SE pooled ) was

calculated by taking the pooled sample variance across all16 chromosomes (as measured
10
by the mean-squared error), dividing by n = 2, and taking the square-root. A critical

value of 0.0016 was used for the t-statistic to correct for the 16 chromosomal
comparisons made per genome (Dunn- idák correction; Sokal and Rohlf ); 95% of such
rejection regions would contain all chromosomes under the null hypothesis of euploidy.
A sequential method (Rice 1989) was used to detect aneuploid chromosomes,
15
whereby we eliminated the data from the chromosome exhibiting the greatest evidence
for aneuploidy, adjusted the fluorescence ratios and the t-statistic, and reevaluated the
remaining chromosomes until none met our criteria for aneuploidy.
References
20
Davis, R.W., M. Thomas, J. Cameron, T.P.S. John, S. Scherer, R.A. Padgett (1980)
Rapid DNA isolation for enzymatic and hybridization analysis. Methods Enzymol
65:404-411
Gerstein, A.C., H.E. Chun, A. Grant, S.P. Otto (2006). Genomic convergence toward
diploidy in Saccharomyces cerevisiae. PLoS Genetics 2: e145
25
Rice W. R. 1989. Analyzing tables of statistical tests. Evolution. 43: 223–225.
Sokal, R. R. and F. J. Rohlf. 1995. Biometry: the principles and practice of statistics in
biological research. 3rd edition. W. H. Freeman and Co. New York.
2