Answers.Body: <p>You can't (easily) use a single design, since clone is nested within diabetic status, so you were correct using two separate designs. Your designs are correct.</p>
  
  <p>If you really wanted to use a single design for some reason, it'd be <code>~clone+structure</code> and for the latter comparison you'd end up using a rather complicated contrast (something like <code>c(rep(c(1,-1), 3), 0)</code>). I really can't recommend that unless you have a very compelling reason, since it's vastly easier to think in terms of factors for your experiment and you're not hurting anything by fitting twice.</p>
  
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Answers.CreationDate: 2019-08-06T10:45:59.143Z
Answers.Text: thank you for the feedback.
Answers.UserDisplayName: mario rossi
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2019-08-05T20:42:37.723Z
Answers.LastActivityDate: 2019-08-05T20:42:37.723Z
Answers.UserDisplayName: Devon Ryan
Body: <p>I am not sure I am building the proper design formula for the question I want to answer</p>
  
  <p>I have the following samples with three factors; clone, the structure and the condition.</p>
  
  <pre><code>clone structure diabetic
  1 07        2D      Dia
  2 21        2D     Ctrl
  3 23        2D      Dia
  4 32        2D     Ctrl
  5 34        2D      Dia
  6 43        2D     Ctrl
  7 07        3D      Dia
  8 21        3D     Ctrl
  9 23        3D      Dia
  10 32        3D     Ctrl
  11 34        3D      Dia
  12 43        3D     Ctrl
  </code></pre>
  
  <p>I want to pull the differentially expressed genes for structure (2D vs 3D) blocking by clone, and the one differentially expressed by condition 
   (regardless of the structure variable).</p>
  
  <p>I am not sure how to build the design in one call, therefore I have produced the first object to pull 2D vs 3D blocking by clone using the following.</p>
  
  <pre><code>des_structure &lt;- DESeqDataSetFromHTSeqCount(sampleTable = samples,
                                         directory = "../data/htseq_geneCounts/",
                                         design= ~ clone+structure)
  </code></pre>
  
  <p>for the condition controlled by structure I have used</p>
  
  <pre><code>des_condidtion &lt;- ddsHTSeq_diabetic &lt;- DESeqDataSetFromHTSeqCount(sampleTable = samples,
                                                   directory = "../data/htseq_geneCounts/",
                                                   design= ~ structure+diabetic)
  </code></pre>
  
  <p>I am not sure whether this is the best way to build the designs.</p>
  
Tags: <rna-seq><deseq2><design>
Title: design formula question
ThreadText: # design formula question
  
  (Asked by: username_0 on 2019-08-05)
  
  I am not sure I am building the proper design formula for the question I want to answer
  
  I have the following samples with three factors; clone, the structure and the condition.
  
  ```
  clone structure diabetic
  1 07        2D      Dia
  2 21        2D     Ctrl
  3 23        2D      Dia
  4 32        2D     Ctrl
  5 34        2D      Dia
  6 43        2D     Ctrl
  7 07        3D      Dia
  8 21        3D     Ctrl
  9 23        3D      Dia
  10 32        3D     Ctrl
  11 34        3D      Dia
  12 43        3D     Ctrl
  
  ```
  
  I want to pull the differentially expressed genes for structure (2D vs 3D) blocking by clone, and the one differentially expressed by condition
  (regardless of the structure variable).
  
  I am not sure how to build the design in one call, therefore I have produced the first object to pull 2D vs 3D blocking by clone using the following.
  
  ```
  des_structure <- DESeqDataSetFromHTSeqCount(sampleTable = samples,
                                         directory = "../data/htseq_geneCounts/",
                                         design= ~ clone+structure)
  
  ```
  
  for the condition controlled by structure I have used
  
  ```
  des_condidtion <- ddsHTSeq_diabetic <- DESeqDataSetFromHTSeqCount(sampleTable = samples,
                                                   directory = "../data/htseq_geneCounts/",
                                                   design= ~ structure+diabetic)
  
  ```
  
  I am not sure whether this is the best way to build the designs.
  
  **Tags:** rna-seq, deseq2, design
  
  
  ---
  
  ## 1 answers
  
  ### Answer by username_1 on 2019-08-05. Score: 6 | Accepted answer
  
  You can't (easily) use a single design, since clone is nested within diabetic status, so you were correct using two separate designs. Your designs are correct.
  
  If you really wanted to use a single design for some reason, it'd be `~clone+structure` and for the latter comparison you'd end up using a rather complicated contrast (something like `c(rep(c(1,-1), 3), 0)`). I really can't recommend that unless you have a very compelling reason, since it's vastly easier to think in terms of factors for your experiment and you're not hurting anything by fitting twice.
  
  > **Comments:**
  > **username_0**: thank you for the feedback.  
  
  ---
  
  