Answers.Body: <p>Leave-one-out cross validation is an iterative method that leaves out one sample until each sample has been left out once. You can then identify the intersection of the differentially expressed genes from the sample space of repeated analyses to produce a set of genes that are consistently differentially expressed independent of individual sample biases.
  more info here:
  <a href="http://efavdb.com/leave-one-out-cross-validation/" rel="nofollow noreferrer">http://efavdb.com/leave-one-out-cross-validation/</a></p>
  
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Answers.CreationDate: 2020-02-09T17:52:33.580Z
Answers.Text: Thanks a lot Drew J., this seems a valid approach! However, I do wonder why there is not a DE method addressing this issue directly. I am not a statistician, so I may have missed some theory behind this, though.
Answers.UserDisplayName: Charles
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-03-02T15:07:15.277Z
Answers.Text: The statistical models generally used for DE involve population level assumptions that are applied to incredibly small sample sizes. The main problem is that sequencing is still prohibitively expensive for small labs to produce data from experiments that include sufficient numbers to address effect size and power.
Answers.UserDisplayName: Andrew Judell
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Answers.CreationDate: 2020-03-02T15:22:50.917Z
Answers.Text: @Charles if you found the answer useful, please up-vote it. I am trying to build up my reputation points on this forum specifically to enable voting. Thank you.
Answers.UserDisplayName: Andrew Judell
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2020-02-06T06:59:33.553Z
Answers.LastActivityDate: 2020-02-06T06:59:33.553Z
Answers.UserDisplayName: Andrew Judell
Answers.Body: <p>One potential problem here is that, as far as I can see, you're not taking the hierarchical structure of your scRNA-seq data into account. I.e. the fact that cells from the same patient / sample will be more similar than cells from different samples, even within the same group. This is also the problem addressed in the paper mentioned by @alan. Also, it usually makes more sense to do this kind of group vs. group comparison <strong>for each subpopulation (cluster/cell type) separately</strong> (assuming you have multiple subpopulations in your data and did some kind of clustering).</p>
  
  <p>One way of doing this is by using a <strong>pseudobulk-level approach</strong>, where you basically aggregate (by either averaging or summing) your data over each sample, for each cluster separately. So you end up with one counts matrix per subpopulation with one row for each gene and one column for each <strong>sample</strong>. The sample columns are then the sums (or averages) of all cells belonging to that cluster-sample combination.</p>
  
  <p>You can then use bulk-RNAseq methods such as <strong>edgeR</strong> or <strong>DESeq2</strong> on the aggregated data to test differences between groups of samples for each cluster. The <a href="https://bioconductor.org/packages/release/bioc/html/muscat.html" rel="nofollow noreferrer">muscat</a> package implements all these steps and has quite extensive documentation. So you could check that and test it out on your data.</p>
  
  <p>The paper describing the method of <em>muscat</em> can be found here: <a href="https://www.biorxiv.org/content/10.1101/713412v2" rel="nofollow noreferrer">https://www.biorxiv.org/content/10.1101/713412v2</a></p>
  
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Answers.CreationDate: 2020-05-11T11:42:01.077Z
Answers.LastActivityDate: 2020-05-11T11:42:01.077Z
Answers.UserDisplayName: milanmlft
Body: <p>I am analysing a human single cell RNA seq experiment, where we have 4 groups, four samples each. Data has been analysed using Seurat, with the canonical workflow. I have tried DE using various methods (Wilcox, MAST, LR), and for each of them the top genes (biggest fold change, lowest adjusted p) are genes over expressed by specific samples, e.g.:
  <a href="https://i.sstatic.net/PCyTH.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/PCyTH.png" alt="enter image description here"></a></p>
  
  <p>Looking at the average expression for these genes per each subject, and plotting on a Boxplot, it does not seem to be a genuine difference (e.g. for the gene above):
  <a href="https://i.sstatic.net/l0041.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/l0041.png" alt="enter image description here"></a></p>
  
  <p>Besides the subject, there is no other parameter (e.g. age, sex) that is biased across groups and that shows up as differentially represented in the PCA plot.</p>
  
  <p>Is there a DE method that takes into account this behaviour and corrects for it?
  How can these results be presented? 
  Besides increasing number of subjects (not really feasible), is there a solution to analyse these data?</p>
  
  <p>Thanks a lot!</p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-02-03T18:07:11.087Z
Comments.Text: How did you scale the data (`ScaleData` or `SCTransform`)? You can usually regress out any source of variation in your data using `vars.to.regress = "Group"`. In your case, "Group" would be the column of `object@metadata` containing the Group information.
Comments.UserDisplayName: fra
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-02-03T18:09:56.770Z
Comments.Text: Thanks for the reply, I did not do SCTransform, and with ScaleData I did not regress any variable. I do not think that scaling by "Group" will be useful: 1) I do not want to mask genuine changes between groups, 2) the DE methods use normalised data, not scaled data.
Comments.UserDisplayName: Charles
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-02-06T10:32:53.580Z
Comments.Text: Regressing out group would eliminate most if not all differential expression between groups. Regressing out sample would be a slightly more reasonable approach but would likely end up being pretty similar
Comments.UserDisplayName: alan ocallaghan
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-02-06T10:33:49.947Z
Comments.Text: This paper might be interesting to you https://www.biorxiv.org/content/10.1101/2020.01.15.906248v1
Comments.UserDisplayName: alan ocallaghan
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-02-09T17:42:55.290Z
Comments.Text: Thanks for the paper @alanocallaghan!
Comments.UserDisplayName: Charles
Tags: <rna-seq><scrnaseq><seurat><differential-expression>
Title: Differential gene expression bias due to effect of an individual sample
ThreadText: # Differential gene expression bias due to effect of an individual sample
  
  (Asked by: username_0 on 2020-02-03)
  
  I am analysing a human single cell RNA seq experiment, where we have 4 groups, four samples each. Data has been analysed using Seurat, with the canonical workflow. I have tried DE using various methods (Wilcox, MAST, LR), and for each of them the top genes (biggest fold change, lowest adjusted p) are genes over expressed by specific samples, e.g.:
  [![enter image description here](https://i.sstatic.net/PCyTH.png)](https://i.sstatic.net/PCyTH.png)
  
  Looking at the average expression for these genes per each subject, and plotting on a Boxplot, it does not seem to be a genuine difference (e.g. for the gene above):
  [![enter image description here](https://i.sstatic.net/l0041.png)](https://i.sstatic.net/l0041.png)
  
  Besides the subject, there is no other parameter (e.g. age, sex) that is biased across groups and that shows up as differentially represented in the PCA plot.
  
  Is there a DE method that takes into account this behaviour and corrects for it?
  How can these results be presented?
  Besides increasing number of subjects (not really feasible), is there a solution to analyse these data?
  
  Thanks a lot!
  
  **Tags:** rna-seq, scrnaseq, seurat, differential-expression
  
  > **Comments:**
  > **username_1**: How did you scale the data (`ScaleData` or `SCTransform`)? You can usually regress out any source of variation in your data using `vars.to.regress = "Group"`. In your case, "Group" would be the column of `object@metadata` containing the Group information.  
  > **username_0**: Thanks for the reply, I did not do SCTransform, and with ScaleData I did not regress any variable. I do not think that scaling by "Group" will be useful: 1) I do not want to mask genuine changes between groups, 2) the DE methods use normalised data, not scaled data.  
  > **username_2**: Regressing out group would eliminate most if not all differential expression between groups. Regressing out sample would be a slightly more reasonable approach but would likely end up being pretty similar  
  > **username_2**: This paper might be interesting to you https://www.biorxiv.org/content/10.1101/2020.01.15.906248v1  
  > **username_0**: Thanks for the paper @username_2!  
  
  ---
  
  ## 2 answers
  
  ### Answer by username_3 on 2020-02-06. Score: 3
  
  Leave-one-out cross validation is an iterative method that leaves out one sample until each sample has been left out once. You can then identify the intersection of the differentially expressed genes from the sample space of repeated analyses to produce a set of genes that are consistently differentially expressed independent of individual sample biases.
  more info here:
  <http://efavdb.com/leave-one-out-cross-validation/>
  
  > **Comments:**
  > **username_0**: Thanks a lot Drew J., this seems a valid approach! However, I do wonder why there is not a DE method addressing this issue directly. I am not a statistician, so I may have missed some theory behind this, though.  
  > **username_3**: The statistical models generally used for DE involve population level assumptions that are applied to incredibly small sample sizes. The main problem is that sequencing is still prohibitively expensive for small labs to produce data from experiments that include sufficient numbers to address effect size and power.  
  > **username_3**: @username_0 if you found the answer useful, please up-vote it. I am trying to build up my reputation points on this forum specifically to enable voting. Thank you.  
  
  ---
  
  ### Answer by username_4 on 2020-05-11. Score: 2
  
  One potential problem here is that, as far as I can see, you're not taking the hierarchical structure of your scRNA-seq data into account. I.e. the fact that cells from the same patient / sample will be more similar than cells from different samples, even within the same group. This is also the problem addressed in the paper mentioned by @username_2. Also, it usually makes more sense to do this kind of group vs. group comparison **for each subpopulation (cluster/cell type) separately** (assuming you have multiple subpopulations in your data and did some kind of clustering).
  
  One way of doing this is by using a **pseudobulk-level approach**, where you basically aggregate (by either averaging or summing) your data over each sample, for each cluster separately. So you end up with one counts matrix per subpopulation with one row for each gene and one column for each **sample**. The sample columns are then the sums (or averages) of all cells belonging to that cluster-sample combination.
  
  You can then use bulk-RNAseq methods such as **edgeR** or **DESeq2** on the aggregated data to test differences between groups of samples for each cluster. The [muscat](https://bioconductor.org/packages/release/bioc/html/muscat.html) package implements all these steps and has quite extensive documentation. So you could check that and test it out on your data.
  
  The paper describing the method of *muscat* can be found here: <https://www.biorxiv.org/content/10.1101/713412v2>
  
  
  ---
  
  