Single-cell eQTL mapping in yeast reveals a tradeoff between growth and reproduction

Reviewer #1 (Public Review):

Summary:

The authors demonstrate that it is possible to carry out eQTL experiments for the model eukaryote S. cerevisiae, in "one pot" preparations, by using single-cell sequencing technologies to simultaneously genotype and measure expression. This is a very appealing approach for investigators studying genetic variation in single-celled and other microbial systems, and will likely inspire similar approaches in non-microbial systems where comparable cell mixtures of genetically heterogeneous individuals could be achieved.

Strengths:

While eQTL experiments have been done for nearly two decades (the corresponding author's lab are pioneers in this field), this single-cell approach creates the possibility for new insights about cell biology that would be extremely challenging to infer using bulk sequencing approaches. The major motivating application shown here is to discover cell occupancy QTL, i.e. loci where genetic variation contributes to differences in the relative occupancy of different cell cycle stages. The authors dissect and validate one such cell cycle occupancy QTL, involving the gene GPA1, a G-protein subunit that plays a role in regulating the mating response MAPK pathway. They show that variation at GPA1 is associated with proportional differences in the fraction of cells in the G1 stage of the cell cycle. Furthermore, they show that this bias is associated with differences in mating efficiency.

Weaknesses:

While the experimental validation of the role of GPA1 variation is well done, the novel cell cycle occupancy QTL aspect of the study is somewhat underexploited. The cell occupancy QTLs that are mentioned all involve loci that the authors have identified in prior studies that involved the same yeast crosses used here. It would be interesting to know what new insights, besides the "usual suspects", the analysis reveals. For example, in Cross B there is another large effect cell occupancy QTL on Chr XI that affects the G1/S stage. What candidate genes and alleles are at this locus? And since cell cycle stages are not biologically independent (a delay in G1, could have a knock-on effect on the frequency of cells with that genotype in G1/S), it would seem important to consider the set of QTLs in concert.

Reviewer #2 (Public Review):

Boocock and colleagues present an approach whereby eQTL analysis can be carried out by scRNA-Seq alone, in a one-pot-shot experiment, due to genotypes being able to be inferred from SNPs identified in RNA-Seq reads. This approach obviates the need to isolate individual spores, genotype them separately by low-coverage sequencing, and then perform RNA-Seq on each spore separately. This is a substantial advance and opens up the possibility to straightforwardly identify eQTLs over many conditions in a cost-efficient manner. Overall, I found the paper to be well-written and well-motivated, and have no issues with either the methodological/analytical approach (though eQTL analysis is not my expertise), or with the manuscript's conclusions.

I do have several questions/comments.

393 segregant experiment:
For the experiment with the 393 previously genotyped segregants, did the authors examine whether averaging the expression by genotype for single cells gave expression profiles similar to the bulk RNA-Seq data generated from those genotypes? Also, is it possible (and maybe not, due to the asynchronous nature of the cell culture) to use the expression data to aid in genotyping for those cells whose genotypes are ambiguous? I presume it might be if one has a sufficient number of cells for each genotype, though, for the subsequent one-pot experiments, this is a moot point.

Figure 1B:
Is UMAP necessary to observe an ellipse/circle - I wouldn't be surprised if a simple PCA would have sufficed, and given the current discussion about whether UMAP is ever appropriate for interpreting scRNA-Seq (or ancestry) data, it seems the PCA would be a preferable approach. I would expect that the periodic elements are contained in 2 of the first 3 principal components. Also, it would be nice if there were a supplementary figure similar to Figure 4 of Macosko et al (PMID 26000488) to indeed show the cell cycle dependent expression.

Aging, growth rate, and bet-hedging:
The mention of bet-hedging reminded me of Levy et al (PMID 22589700), where they saw that Tsl1 expression changed as cells aged and that this impacted a cell's ability to survive heat stress. This bet-hedging strategy meant that the older, slower-growing cells were more likely to survive, so I wondered a couple of things. It is possible from single-cell data to identify either an aging, or a growth rate signature? A number of papers from David Botstein's group culminated in a paper that showed that they could use a gene expression signature to predict instantaneous growth rate (PMID 19119411) and I wondered if a) this is possible from single-cell data, and b) whether in the slower growing cells, they see markers of aging, whether these two signatures might impact the ability to detect eQTLs, and if they are detected, whether they could in some way be accounted for to improve detection.

AIL vs. F2 segregants:
I'm curious if the authors have given thought to the trade-offs of developing advanced intercross lines for scRNA-Seq eQTL analysis. My impression is that AIL provides better mapping resolution, but at the expense of having to generate the lines. It might be useful to see some discussion on that.

10x vs SPLit-Seq
10x is a well established, but fairly expensive approach for scRNA-Seq - I wondered how the cost of the 10x approach compares to the previously used approach of genotyping segregants and performing bulk RNA-Seq, and how those costs would change if one used SPLiT-Seq (see PMID 38282330).

Author Response

Provisional response

We would like to thank the reviewers for taking the time to review our manuscript, for providing useful suggestions for improvement, and for highlighting the significance of our approach.

Reviewer #1 (Public Review):

Summary:

The authors demonstrate that it is possible to carry out eQTL experiments for the model eukaryote S. cerevisiae, in "one pot" preparations, by using single-cell sequencing technologies to simultaneously genotype and measure expression. This is a very appealing approach for investigators studying genetic variation in single-celled and other microbial systems, and will likely inspire similar approaches in non-microbial systems where comparable cell mixtures of genetically heterogeneous individuals could be achieved.

Strengths:

While eQTL experiments have been done for nearly two decades (the corresponding author's lab are pioneers in this field), this single-cell approach creates the possibility for new insights about cell biology that would be extremely challenging to infer using bulk sequencing approaches. The major motivating application shown here is to discover cell occupancy QTL, i.e. loci where genetic variation contributes to differences in the relative occupancy of different cell cycle stages. The authors dissect and validate one such cell cycle occupancy QTL, involving the gene GPA1, a G-protein subunit that plays a role in regulating the mating response MAPK pathway. They show that variation at GPA1 is associated with proportional differences in the fraction of cells in the G1 stage of the cell cycle. Furthermore, they show that this bias is associated with differences in mating efficiency.

We thank the reviewer for recognizing the strengths of our overall approach and our dissection of the functional consequences of the W82R variant of GPA1.

Weaknesses:

While the experimental validation of the role of GPA1 variation is well done, the novel cell cycle occupancy QTL aspect of the study is somewhat underexploited. The cell occupancy QTLs that are mentioned all involve loci that the authors have identified in prior studies that involved the same yeast crosses used here. It would be interesting to know what new insights, besides the "usual suspects", the analysis reveals. For example, in Cross B there is another large effect cell occupancy QTL on Chr XI that affects the G1/S stage. What candidate genes and alleles are at this locus?

We thank the reviewer for this suggestion. We plan to expand the section on cell cycle occupancy QTL in our revision.

And since cell cycle stages are not biologically independent (a delay in G1, could have a knock-on effect on the frequency of cells with that genotype in G1/S), it would seem important to consider the set of QTLs in concert.

We thank the reviewer for this suggested clarification. In our revision, we will clarify that the cell cycle occupancy phenotype represents the proportion of cells assigned to a given stage. As the reviewer correctly notes, a change in the proportion of cells in one stage may alter the proportion of cells in other stages, and this could result in cell cycle occupancy QTL for multiple stages. We will make efforts to consider the cell cycle occupancy QTLs in concert in the revised manuscript.

Reviewer #2 (Public Review):

Boocock and colleagues present an approach whereby eQTL analysis can be carried out by scRNA-Seq alone, in a one-pot-shot experiment, due to genotypes being able to be inferred from SNPs identified in RNA-Seq reads. This approach obviates the need to isolate individual spores, genotype them separately by low-coverage sequencing, and then perform RNA-Seq on each spore separately. This is a substantial advance and opens up the possibility to straightforwardly identify eQTLs over many conditions in a cost-efficient manner. Overall, I found the paper to be well-written and well-motivated, and have no issues with either the methodological/analytical approach (though eQTL analysis is not my expertise), or with the manuscript's conclusions.

We thank the reviewer for recognizing the significant contributions our work makes to the field.

393 segregant experiment:

For the experiment with the 393 previously genotyped segregants, did the authors examine whether averaging the expression by genotype for single cells gave expression profiles similar to the bulk RNA-Seq data generated from those genotypes? Also, is it possible (and maybe not, due to the asynchronous nature of the cell culture) to use the expression data to aid in genotyping for those cells whose genotypes are ambiguous? I presume it might be if one has a sufficient number of cells for each genotype, though, for the subsequent one-pot experiments, this is a moot point.

We thank the reviewer for this comment. While we could expand the analysis along these lines, this is not relevant for the subsequent one-pot eQTL experiments, as the reviewer notes, and is therefore beyond the scope of the manuscript. We will make the data available so that anyone interested can try these analyses.

Figure 1B:

Is UMAP necessary to observe an ellipse/circle - I wouldn't be surprised if a simple PCA would have sufficed, and given the current discussion about whether UMAP is ever appropriate for interpreting scRNA-Seq (or ancestry) data, it seems the PCA would be a preferable approach. I would expect that the periodic elements are contained in 2 of the first 3 principal components. Also, it would be nice if there were a supplementary figure similar to Figure 4 of Macosko et al (PMID 26000488) to indeed show the cell cycle dependent expression.

We thank the reviewer for this comment. We too have been following the debate on the utility of UMAP for scRNA-seq, and in our revision we will provide an alternative visualization of the cell cycle. We will also generate a supplementary figure similar to Figure 4 of Macosko et al. to visualize cell-cycle-dependent gene expression.

Aging, growth rate, and bet-hedging:

The mention of bet-hedging reminded me of Levy et al (PMID 22589700), where they saw that Tsl1 expression changed as cells aged and that this impacted a cell's ability to survive heat stress. This bet-hedging strategy meant that the older, slower-growing cells were more likely to survive, so I wondered a couple of things. It is possible from single-cell data to identify either an aging, or a growth rate signature? A number of papers from David Botstein's group culminated in a paper that showed that they could use a gene expression signature to predict instantaneous growth rate (PMID 19119411) and I wondered if a) this is possible from single-cell data, and b) whether in the slower growing cells, they see markers of aging, whether these two signatures might impact the ability to detect eQTLs, and if they are detected, whether they could in some way be accounted for to improve detection.

We thank the reviewer for this comment and suggested analyses. We are not sure whether one can see gene expression signatures of aging in yeast scRNA-seq data. We believe that such analyses are beyond the scope of this work, but we will make the data available so that anyone interested can try them.

AIL vs. F2 segregants:

I'm curious if the authors have given thought to the trade-offs of developing advanced intercross lines for scRNA-Seq eQTL analysis. My impression is that AIL provides better mapping resolution, but at the expense of having to generate the lines. It might be useful to see some discussion on that.

We thank the reviewer for their comment. We will include some discussion of the trade-offs of different experimental designs in our revision.

10x vs SPLit-Seq

10x is a well established, but fairly expensive approach for scRNA-Seq - I wondered how the cost of the 10x approach compares to the previously used approach of genotyping segregants and performing bulk RNA-Seq, and how those costs would change if one used SPLiT-Seq (see PMID 38282330).

We will provide some ballpark estimates of the costs, and we will discuss the trade-offs of different scRNA-seq technologies in our revision