Research article                                                                                Genomics and Evolutionary Biology
Table 2. Type of mutation and AT bias found in ura4 and ura5 mutants at various time points of
quiescence.
day 1          day 8            day 15             day 8+15
# of SNVs                                   105            107              37                 144
Ts                                          33             26               5                  30
Tv                                          72             81               34                 116
Ts:Tv                                       1:2.18         1:3.12           1:6.67             1:3.70
To A or to T                                63             45               15                 60
To C or to G                                17             30               8                  38
AT bias                                     3.71           1.50             1.88               1.58
# of indels                                 40             139              86                 220
# of deletions                              16             99               71                 170
lost bases                                  334            864              907                1771
# of insertions                             24             35               15                 50
gained bases                                269            401              99                 500
net loss                                    65             463              808                1271
# of deletions / # of insertions            0.7            2.8              4.7                3.4
average loss per event                      21             9                13                 10
average gain per event                      11             11               7                  10
Ts: transitions; Tv: transversions
AT bias: (GCfiAT + GCfiTA) to (ATfiGC + ATfiCG) mutations
DOI: https://doi.org/10.7554/eLife.27469.007
observed with a net gain of 269 bp for 24 events and loss of 334 bp for 16 events (Table 2), includ-
ing two deletions of 165 and 95 base-pairs (Supplementary files 1). Overall, the mutation profile at
day one is similar to published results in cycling cells for URA3 in budding yeast (Lang and Murray,
2008) and for ura4+ and ura5+ in fission yeast (Fraser et al., 2003).
During quiescence, the total number of mutations (including redundant ones) resulting in FOAR
colonies increases linearly as a function of time. From multiple experiments, we used least squares
regression to determine that the slope is 0.55  10 7 FOAR mutants per day spent in quiescence
(Figure 2A). Importantly, we observed that the number of redundant mutations dramatically fades
over time, indicating that novel mutations arise in quiescence (Supplementary files 1). Days 8 and
15 are not statistically different for the Ts:Tv and AT bias (chi2 test) and were therefore combined.
Day 1 is statistically different from days 8 + 15 (Ts/Tv: p<0.05; AT bias: p<0.05 by chi2 tests). The
ratio of transition-to-transversion observed at days 8 + 15 of quiescence was reduced from 1:2.18 to
1:3.7 and the mutational bias toward A/T decreased (3.71 vs 1.58, Table 1). This is mainly due to a
relative decrease of the G:C to A:T transitions that is balanced by an increase of the A:T to C:G and
G:C to C:G transversions (Figure 2B and Figure 2—figure supplement 1). During two weeks of qui-
escence, the proportion of de novo indels increases from 28% at day 1 to 57% and 69% after 8 and
15 days, respectively (Figure 2C) to outnumber the SNVs (Figure 2C and D) (day 1 vs days 8 + 15:
p<10 10, chi2 test). Altogether, we found more deletions than insertions (in days 8 + 15: p<10 5,
chi2 test) with a net loss of 1771 bp in 170 mutants and a gain of 500 bp in 50 mutants (Figure 2D,
Table 2). The main class of indels is ±1 bp and accounts for roughly half the events (Figure 2D).
More than one half of the indels occur within homonucleotide runs and several mutations are com-
plex (Supplementary files 1). Collectively, we found a phenotypic mutation rate of 0.55  10 7
FOAR colonies per day of quiescence with 0.14  10 7 FOAR colonies per day due to SNVs and 0.41
10 7 FOAR colonies per day due to indels (Figure 3A,B). Thus, the mutational spectra of growth
and quiescence exhibit striking quantitative and qualitative differences. First, elevated levels of
indels are generated. Second, two recent MA studies (Behringer and Hall, 2015; Farlow et al.,
2015) with fission yeast have shown that in cycling cells insertions outnumber deletions, whereas we
observe the reverse in quiescence (Figure 2D). Thus, growth and quiescence apply opposite
Gangloff et al. eLife 2017;6:e27469. DOI: https://doi.org/10.7554/eLife.27469                                                           5 of 19