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 | Figure 1 : Survival and physiological
parameters for heterogeneous mutant populations during starvation
conditions. The entire collection of haploid deletion mutants was
starved for either phosphate (+) or leucine (x) in the presence (solid
line) or absence (dashed line) of kanamycin for nearly 500 hours
following an initial period of 24 hours of batch growth. (A) Survival
of replicate populations grown in media of identical composition
except for the limiting nutrient. Survival of each population was
monitored by determining viability of the population at each time
point by counting CFUs on rich media plates. (B) The total number of
cells/mL was determined at each time point and remained essentially
unchanged throughout the starvation regime. (C) Culture biomass was
estimated using a Klett colorimeter and showed a gradual increase for
populations starved for phosphate and gradual decrease when
populations were starved for leucine. (D) The average cell volume,
measured using a Coulter counter, showed a gradual increase for
populations starved for phosphate and a slow decline for populations
starved for leucine. |
 | Figure 2 : Development and validation
of quantitative barcode sequencing for multiplexed mutant screens. We
tested the error and variance associated with each step of our
protocol. . (A) By sequencing a single barcode we found that 98% of
sequences up to sequencing cycle 23 (gray dotted line) perfectly match
the expected sequence (n = 2,340,984). (B) Additional cycles of PCR
introduce minimal variation in the estimated proportions of mutants.
The best-correlated estimates of mutant abundance are found between
15-25 PCR cycles (increasingly black values approach a correlation of
1.0; the minimum correlation is 0.94). (C) Resequencing the same PCR
product from a complex mixture of mutants on two different flow cells
yields highly reproducible results (Pearson correlation = 0.99; n =
3,329). (D) Complete technical replicates of quantitative barcode
sequencing (i.e. independent DNA preparations, PCR and sequencing
reactions) are highly reproducible (Pearson correlation = 0.94; n =
3,439). |
 |
Figure 3 : Experimental design
for multiplexed mutant survival analysis using quantitative barcode
sequencing. We constructed normalized pools of the yeast haploid
deletion collection by growing individual mutants on rich media (YDP)
plates and pooling mutants in liquid YPD for archival purposes. A
1.6mL aliquot of the unselected pooled mutants was used to inoculate
(t=0) cultures limited for either phosphate or leucine. The
starvation period commenced after 24 hours of culture growth. At each
time point we removed a 1mL sample from the culture and expanded the
viable sub-population by allowing 24 hours of outgrowth in
supplemented minimal media. DNA was isolated from the resulting
culture and analyzed using quantitative barcode sequencing. |
 | Figure 4 : Population diversity decline
and mutant abundance profiles during prolonged starvation. (A) We
determined the number of unique strains identified through barcode
sequencing at each time point for populations starved for phosphate
(gray bars) or leucine (black bars). (B) Hierarchical clustering of
mutant abundance profiles during starvation experiments. We clustered
vectors of relative abundance in the population normalized by the
abundance of each mutant at t = 24 hours (log2 transformed). Black
indicates that the strain has not changed in abundance. Yellow
represents increases in abundance and blue represents decreases in
abundance. Failure to detect the strain in the population is
indicated by gray. We identified clusters of mutants that were either
specifically (C) decreased in relative abundance upon phosphate
starvation or (D) increased in relative abundance upon leucine
starvation. Several mutants are decreased in relative abundance under
both starvation conditions including (E) a cluster including several
autophagy gene mutants. |
 | Figure 5 : Quantitative analysis of
absolute death rates during prolonged starvation. We calculated the
absolute rate of death for mutants using measurements of population
viability and estimates of relative strain abundance using
quantitative barcode sequencing of uptags (circles) and downtags
(triangles) for the putatively neutral HOÄ0 allele starved for
phosphate (A) and leucine (B). The data presented are barcode counts
normalized between all uptag or downtag sequencing results. A value
of 1 was added to all normalized barcode counts prior to log2
transformation. The rate of death was determined using a generalized
linear model for uptag (long dash line) and downtag (short dash line)
data. These rates were compared to rates calculated from independent
data (viable cells / ìL)obtained for the isogenic strain BY4742
(crosses) subjected to starvation in pure cultures. We calculated
death rates for all mutants in each starvation condition and converted
these values to half-lives for all barcode data that yielded a
significant death rate (FDR <5%). The distribution of half-lives for
mutants starved for phosphate is centered around 289 hours (C) and
22.8 hours (D) for leucine starved mutants. The half-life of the HOÄ0
strain when starved for phosphate (blue dotted line) or leucine (red
dotted line in both histograms) is shown for reference. |
 | Figure 6 : Functional gene modules
altering survival during nutrient starvation. The distributions of
half-lives for subsets of genes defined by different methods of
categorization were compared with the overall distribution of
half-lives (white bars) in each experiment shown. (A) Phosphate
starvation, mitochondrion organization (blue, Go Slim term, n = 281, p
= 5.88 x 10-41)), peroxisome organization (red, GO:0007031, n = 27, p
= 0.00013) (B) Leucine starvation, mitochondrion organization (blue,
Go Slim term, n = 276, p = 9.23 x 10-14), peroxisome organization
(red, GO:0007031, n = 25, p = 4.63 x10-5) (C) Phosphate starvation,
autophagy (blue, GO:0006914, n = 51, p = 2.0 x 10-13), translation
(red, GO:0006412, n = 305, p = 1.46 x 10-12) (D) Leucine starvation,
autophagy (blue, GO:0006914, n = 50, p = 0.00014), translation (red,
GO:0006412, n = 301, p = 2.33 x 10-11) (E) Phosphate starvation, mRNA
processing (blue, GO:0006397, n = 91, p = 3.38 x 10-11), mRNA
transport (red, GO:0051028, n = 42, p = 6.45 x 10-5) (F) Leucine
starvation, mRNA processing (blue, GO:0006397, n = 91, p = 0.87), mRNA
transport (red, GO:0051028, n = 40, p = 0.87) (G) Phosphate
starvation, chromatin modification (blue, GO:0016568, n = 89, p = 2.86
x 10-6), histone acetyltransferase complex (red, SGD defined protein
complex, n = 41, p = 8.82 x 10-5) (H) Leucine starvation, chromatin
modification (blue, GO:0016568, n = 90, p = 0.032), histone
acetyltransferase complex (red, SGD defined protein complex, n = 40, p
= 0.16) (I) Phosphate starvation, cytoskeleton (blue, GO:0005856, n =
151, p = 2.03 x 10-5), microtubule organizing center (red, SGD defined
protein complex, n = 30, p = 3.16 x 10-5) (J) Leucine starvation,
cytoskeleton (blue, GO:0005856, n = 148, p = 0.14), microtubule
organizing center (red, SGD defined protein complex, n = 30, p = 0.75)
(K) Phosphate starvation, slow growth in YPD (blue, defined by
(GIAEVER et al. 2002), n = 637, p =2.46 x 10-103), impaired growth in
rapamycin (red, defined by (DUDLEY et al. 2005), n = 137, p = 1.16 x
10-19) (L) Leucine starvation, slow growth in YPD (blue, defined by
(GIAEVER et al. 2002), n = 619, p = 3.04 x 10-16), impaired growth in
rapamycin (red, defined by (DUDLEY et al. 2005), n = 132, p = 0.00058)
(M) Phosphate starvation, MRPL10 cluster of the yeast metabolic cycle
(blue, defined in (TU et al. 2005), n = 54, p = 1.62 x 10-16) (N)
Leucine starvation, MRPL10 cluster of the yeast metabolic cycle (blue,
defined in (TU et al. 2005), n = 53, p = 4.8 x 10-6). |
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