Answers.Body: <p>Mike Love is right. <strong>If</strong> the response you are looking for is linear in terms of change per minute, the most productive approach is likely to be fitting a linear model. You <strong>might</strong> get something out of this because the difference between successive time points represents multiple measurements of the rate of change over time. The biggest problem is that the time points aren't independent. You would need to fit a model that estimated that rate for both WT and KO and then test the difference in rates.</p>
  
  <p>I see three ways forward, in all cases you want to test the last term in the model <code>~time + genotype + time:genotype</code></p>
  
  <ol>
  <li><p>Ideally you would like to fit a negative binomial linear model to the read counts. But that is going to be difficult as you have no replicates from which to assess the dispersion. Thus you could generate read counts using your favorite read count pipeline (STAR+featureCounts/Salmon/Kallisto) and take the approach in the edgeR guide noted by <a href="https://bioinformatics.stackexchange.com/a/2144/235" title="b.nota">b.nota</a> and then use edgeR to test the fitted model.</p></li>
  <li><p>You could use something like salmon or kallisto to estimate the TPMs assume that log TPMs are normally distributed and model them with plain <code>lm</code> in R, using something like <code>broom</code> to model each gene separately. A tutorial by David Robinson, the author of <code>broom</code> on doing this sort of thing can be found <a href="http://varianceexplained.org/r/tidy-genomics-broom/" rel="noreferrer" title="here">here</a></p></li>
  <li><p>You could use the Salmon or Kallisto bootstraps to estimate <em>technical</em> variation (but not biological) and then I suspect you could use Sleuth to fit the model.</p></li>
  </ol>
  
  <p>In any case these results should be treated with scepticism andmostly treated as hypothesis generating. You might find genes that you want to go back and confirm and you might see some patterns of interest if you look at some enrichments etc. which you can then design experiments to test. </p>
  
Answers.ContentLicense: CC BY-SA 3.0
Answers.CreationDate: 2017-07-26T13:53:55.933Z
Answers.Text: thank you for the information is there a tutorial for the lm and broom methods that you have mentioned would help me understand it better, as R is something I have just started using appreciate it.
Answers.UserDisplayName: novicebioinforesearcher
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Answers.Text: I have added a link to a broom tutorial.
Answers.UserDisplayName: Ian Sudbery
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Answers.CreationDate: 2017-07-26T14:53:32.823Z
Answers.Text: it is really an awesome resource many thanks!! (I would not have found this), and keep everyone posted hopefully i can pick up on using R and work this out within a couple of days.
Answers.UserDisplayName: novicebioinforesearcher
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Answers.LastEditDate: 2017-07-26T14:28:53.783Z
Answers.UserDisplayName: Ian Sudbery
Answers.Body: <p>Although it is not recommended to use no replicates, in the edgeR manual they give some advice on how to go on with no replicates design. See page 21 of user <a href="https://bioconductor.org/packages/release/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf" rel="noreferrer">guide</a>. You can e.g. estimate a BCV value. Of course it is still a trick and not sound statistics.</p>
  
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Answers.UserDisplayName: benn
Body: <p>We have an experimental design as seen below </p>
  
  <p>Where we administered drug at 0 min for each mouse genotype and took them down at given below intervals. wt and ko mouse models  were administered only with the vehicle which would be our negative control.</p>
  
  <p>Performed rnaseq (total rna) truseq stranded protocol would like to see differences between each time point compared to the vehicle, used star aligner to align to mm9 , htseq for gene counts . </p>
  
  <pre><code>sample  type    time
  WT  Vehicle     30 min
  KO  Vehicle     30 min
  WT  Drug    30min
  KO  Drug    30min
  WT  Drug    1 hour
  KO  Drug    1 hour
  WT  Drug    2 hour
  KO  Drug    2hour
  WT  Drug    3 hour
  KO  Drug    3hour
  </code></pre>
  
  <p>We do not have any replicates and hence cannot use any of the differential expression statistical tools (asked this question at bioconductor where Dr. Love himself replied <a href="https://support.bioconductor.org/p/98429/#98456" rel="noreferrer">link to question asked</a>) where he suggested a linear model. </p>
  
  <p>we were wondering if someone could help us with any such design (papers/previously performed analysis) what we would like to see is comparison/differences (significant) between these groups 
  (veh vs 30min, veh vs 1 hour, beh vs 2 hours, veh vs 3 hours) </p>
  
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2017-07-25T17:45:11.150Z
Comments.Text: What is your design, exactly? What are we supposed to understand from that vague table? What are the times? The time you administered the drug? The length of time the drug had to act before administering an antagonist? The time you spent observing it? How is this related to bioinformatics? Please [edit] your question and explain what you are doing and what you expect from a good answer.
Comments.UserDisplayName: terdon
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Comments.CreationDate: 2017-07-25T18:01:49.310Z
Comments.Text: It's unlikely that you'll find any papers doing this because it'd be hard to get one accepted if it were largely based on such an experiment. I have to wonder why you expended time/resources on this experiment when you had to know that this was going to be a major problem.
Comments.UserDisplayName: Devon Ryan
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2017-07-25T18:53:20.763Z
Comments.Text: @DevonRyan we have experimental (wet lab results supporting) this, realized it will be difficult to go ahead with rna seq only after i posted it on deseq forum (bioconductor), we are not sure if this is a dead end or we can at least see some basic differences using very basic stats
Comments.UserDisplayName: novicebioinforesearcher
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Comments.CreationDate: 2017-07-25T18:56:40.270Z
Comments.Text: @terdon thank you for feedback i have edited the question, please let me know if further information is required
Comments.UserDisplayName: novicebioinforesearcher
Comments.ContentLicense: CC BY-SA 3.0
Comments.CreationDate: 2017-07-25T19:23:19.073Z
Comments.Text: So you administered the drug and then killed the mice after the specified time. Then what? Did you perform rna seq? Of what? Whole transcriptome? What are you trying to show? What data do you have? Are you really trying to draw conclusions based on a single individual in each condition?
Comments.UserDisplayName: terdon
Tags: <rna-seq><deseq2><differential-expression>
Title: differential gene expression complex design no replicates
ThreadText: # differential gene expression complex design no replicates
  
  (Asked by: username_0 on 2017-07-25)
  
  We have an experimental design as seen below
  
  Where we administered drug at 0 min for each mouse genotype and took them down at given below intervals. wt and ko mouse models were administered only with the vehicle which would be our negative control.
  
  Performed rnaseq (total rna) truseq stranded protocol would like to see differences between each time point compared to the vehicle, used star aligner to align to mm9 , htseq for gene counts .
  
  ```
  sample  type    time
  WT  Vehicle     30 min
  KO  Vehicle     30 min
  WT  Drug    30min
  KO  Drug    30min
  WT  Drug    1 hour
  KO  Drug    1 hour
  WT  Drug    2 hour
  KO  Drug    2hour
  WT  Drug    3 hour
  KO  Drug    3hour
  
  ```
  
  We do not have any replicates and hence cannot use any of the differential expression statistical tools (asked this question at bioconductor where Dr. Love himself replied [link to question asked](https://support.bioconductor.org/p/98429/#98456)) where he suggested a linear model.
  
  we were wondering if someone could help us with any such design (papers/previously performed analysis) what we would like to see is comparison/differences (significant) between these groups
  (veh vs 30min, veh vs 1 hour, beh vs 2 hours, veh vs 3 hours)
  
  **Tags:** rna-seq, deseq2, differential-expression
  
  > **Comments:**
  > **username_1**: What is your design, exactly? What are we supposed to understand from that vague table? What are the times? The time you administered the drug? The length of time the drug had to act before administering an antagonist? The time you spent observing it? How is this related to bioinformatics? Please [edit] your question and explain what you are doing and what you expect from a good answer.  
  > **username_2**: It's unlikely that you'll find any papers doing this because it'd be hard to get one accepted if it were largely based on such an experiment. I have to wonder why you expended time/resources on this experiment when you had to know that this was going to be a major problem.  
  > **username_0**: @username_2 we have experimental (wet lab results supporting) this, realized it will be difficult to go ahead with rna seq only after i posted it on deseq forum (bioconductor), we are not sure if this is a dead end or we can at least see some basic differences using very basic stats  
  > **username_0**: @username_1 thank you for feedback i have edited the question, please let me know if further information is required  
  > **username_1**: So you administered the drug and then killed the mice after the specified time. Then what? Did you perform rna seq? Of what? Whole transcriptome? What are you trying to show? What data do you have? Are you really trying to draw conclusions based on a single individual in each condition?  
  
  ---
  
  ## 2 answers
  
  ### Answer by username_4 on 2017-07-26. Score: 7 | Accepted answer
  
  Although it is not recommended to use no replicates, in the edgeR manual they give some advice on how to go on with no replicates design. See page 21 of user [guide](https://bioconductor.org/packages/release/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf). You can e.g. estimate a BCV value. Of course it is still a trick and not sound statistics.
  
  
  ---
  
  ### Answer by username_3 on 2017-07-26. Score: 7
  
  Mike Love is right. **If** the response you are looking for is linear in terms of change per minute, the most productive approach is likely to be fitting a linear model. You **might** get something out of this because the difference between successive time points represents multiple measurements of the rate of change over time. The biggest problem is that the time points aren't independent. You would need to fit a model that estimated that rate for both WT and KO and then test the difference in rates.
  
  I see three ways forward, in all cases you want to test the last term in the model `~time + genotype + time:genotype`
  
  1. Ideally you would like to fit a negative binomial linear model to the read counts. But that is going to be difficult as you have no replicates from which to assess the dispersion. Thus you could generate read counts using your favorite read count pipeline (STAR+featureCounts/Salmon/Kallisto) and take the approach in the edgeR guide noted by [b.nota](https://bioinformatics.stackexchange.com/a/2144/235 "b.nota") and then use edgeR to test the fitted model.
  2. You could use something like salmon or kallisto to estimate the TPMs assume that log TPMs are normally distributed and model them with plain `lm` in R, using something like `broom` to model each gene separately. A tutorial by David Robinson, the author of `broom` on doing this sort of thing can be found [here](http://varianceexplained.org/r/tidy-genomics-broom/ "here")
  3. You could use the Salmon or Kallisto bootstraps to estimate *technical* variation (but not biological) and then I suspect you could use Sleuth to fit the model.
  
  In any case these results should be treated with scepticism andmostly treated as hypothesis generating. You might find genes that you want to go back and confirm and you might see some patterns of interest if you look at some enrichments etc. which you can then design experiments to test.
  
  > **Comments:**
  > **username_0**: thank you for the information is there a tutorial for the lm and broom methods that you have mentioned would help me understand it better, as R is something I have just started using appreciate it.  
  > **username_3**: I have added a link to a broom tutorial.  
  > **username_0**: it is really an awesome resource many thanks!! (I would not have found this), and keep everyone posted hopefully i can pick up on using R and work this out within a couple of days.  
  
  ---
  
  