Answers.Body: <p>Yes, you can safely concatenate the technical replicates. Odds are good that these are even the same libraries just sequenced twice, so even labeling them as replicates is a bit of a stretch. As an aside, it would be surprising if you actually had a batch effect in a situation like this. You will commonly see sequencing facilities just sequence a given sample a second time if it didn't get the number of reads requested by the client to begin with. They then just have the client concatenate the files.</p>
  
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Answers.CreationDate: 2019-09-15T23:40:28.323Z
Answers.Text: Sorry @Devon, I have 4 lanes for each samples (paired end) in each of experimental runs; For concatenating fastq files can I do like this ?                  cat          fastq1_lane1_batch1 fastq1_lane1_batch2 fastq1_lane2_batch1   fastq1_lane2_batch2  fastq1_lane3_batch1 fastq1_lane3_batch2 fastq1_lane4_batch1 fastq1_lane4_batch2                             fastq2_lane1_batch1 fastq2_lane1_batch2 fastq2_lane2_batch1   fastq2_lane2_batch2  fastq2_lane3_batch1 fastq2_lane3_batch2 fastq2_lane4_batch1 fastq2_lane4_batch2
Answers.UserDisplayName: Zizogolu
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Answers.Text: @Fereshteh That's a different question, and one you can find answers to by searching this site or biostars. Please do not stretch people offering to help by piling on more questions.
Answers.UserDisplayName: Ram RS
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Answers.Text: Sorry @Devon I think this question relates to this post; Can I use technical replicates for gene differential expression analysis? For example I am only interested in tumour A and normal A , can I compare them by using batch1 and batch2 as my replications?
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Answers.Text: I think you know the answer to that is "no".
Answers.UserDisplayName: Devon Ryan
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Answers.UserDisplayName: Devon Ryan
Answers.Body: <p>Prepping the RNA on different days, or making Illumina libraries on different days, or having different technicians handle different samples; that can lead to batch effects. 
   Running samples on two different days does not cause a significant batch effect, as you can plainly see in your PCA.  You should just combine the fastqs.</p>
  
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Answers.Text: Thank you @swbarnes2, now my question is can I compare only one tumour with its own matched normal sample to get differentially expressed genes for this pair by using experimental runs (technical replicates )? Design would be Tumour1_batch1 Tumour1_batch2 versus Normal1_batch1 Normal1_bacth2
Answers.UserDisplayName: Zizogolu
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Answers.Text: @Fereshteh As you're well aware, new questions should be posted as such.
Answers.UserDisplayName: Devon Ryan
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Answers.UserDisplayName: swbarnes2
Body: <p>I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example <code>tumor 1 in batch 1 and tumor 1 in batch 2 , normal 2 in batch1 and normal 2 in batch 2</code>. This is my design for DESeq2</p>
  
  <pre><code>&gt; head(mycols)
          condition batch
  N_1_305         N     1
  N_1_310         N     1
  N_1_337         N     1
  N_1_353         N     1
  T_1_305         T     1
  T_1_310         T     1
  &gt; tail(mycols)
          condition batch
  T_2_337         T     2
  T_2_338         T     2
  T_2_344         T     2
  T_2_346         T     2
  T_2_349         T     2
  T_2_353         T     2
  &gt;
  </code></pre>
  
  <p>I got this PCA plot</p>
  
  <p><a href="https://i.sstatic.net/IYWxj.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/IYWxj.png" alt="enter image description here"></a></p>
  
  <p>And this is biplot of samples
  <a href="https://i.sstatic.net/E2DOe.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/E2DOe.png" alt="enter image description here"></a></p>
  
  <p>In PCA plot I am seeing for instance 
  , <code>T_1_337 (batch1) has been placed too close to T_2_337 (batch2)</code></p>
  
  <p>Then I used svd for detecting hidden batch </p>
  
  <p><a href="https://i.sstatic.net/Ab2uk.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/Ab2uk.png" alt="enter image description here"></a></p>
  
  <p>Does this mean that there is no big batch effect between experimental runs and I can concatenate the fastqs from both batches for each sample (technical replication) or collapse technical replicates afterwards?</p>
  
  <p>Please help me to interpret these
  Thank you</p>
  
  <hr>
  
  <blockquote>
    <p>EDITED</p>
  </blockquote>
  
  <hr>
  
  <p>Sorry @Devon, I have 4 lanes for each samples (paired end) in each of experimental runs; For concatenating fastq files can I do like this ?</p>
  
  <pre><code>cat fastq1_lane1_batch1 fastq1_lane1_batch2 fastq1_lane2_batch1   fastq1_lane2_batch2  fastq1_lane3_batch1 fastq1_lane3_batch2 fastq1_lane4_batch1 fastq1_lane4_batch2  &gt; fastq1  
  
  cat fastq2_lane1_batch1 fastq2_lane1_batch2 fastq2_lane2_batch1   fastq2_lane2_batch2  fastq2_lane3_batch1 fastq2_lane3_batch2 fastq2_lane4_batch1 fastq2_lane4_batch2 &gt; fastq2
  </code></pre>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2019-09-15T20:34:34.793Z
Comments.Text: Why do you want to collapse replicates? It's really good that you have replicates. Treat them as such.
Comments.UserDisplayName: burger
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2019-09-15T21:36:19.930Z
Comments.Text: She's talking about technical replicates.
Comments.UserDisplayName: Devon Ryan
Tags: <r><rna-seq><deseq2><pca><batch-effects>
Title: Interpreting this PCA plot for RNA-seq
ThreadText: # Interpreting this PCA plot for RNA-seq
  
  (Asked by: username_0 on 2019-09-15)
  
  I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example `tumor 1 in batch 1 and tumor 1 in batch 2 , normal 2 in batch1 and normal 2 in batch 2`. This is my design for DESeq2
  
  ```
  > head(mycols)
          condition batch
  N_1_305         N     1
  N_1_310         N     1
  N_1_337         N     1
  N_1_353         N     1
  T_1_305         T     1
  T_1_310         T     1
  > tail(mycols)
          condition batch
  T_2_337         T     2
  T_2_338         T     2
  T_2_344         T     2
  T_2_346         T     2
  T_2_349         T     2
  T_2_353         T     2
  >
  
  ```
  
  I got this PCA plot
  
  [![enter image description here](https://i.sstatic.net/IYWxj.png)](https://i.sstatic.net/IYWxj.png)
  
  And this is biplot of samples
  [![enter image description here](https://i.sstatic.net/E2DOe.png)](https://i.sstatic.net/E2DOe.png)
  
  In PCA plot I am seeing for instance
  , `T_1_337 (batch1) has been placed too close to T_2_337 (batch2)`
  
  Then I used svd for detecting hidden batch
  
  [![enter image description here](https://i.sstatic.net/Ab2uk.png)](https://i.sstatic.net/Ab2uk.png)
  
  Does this mean that there is no big batch effect between experimental runs and I can concatenate the fastqs from both batches for each sample (technical replication) or collapse technical replicates afterwards?
  
  Please help me to interpret these
  Thank you
  
  ---
  
  > EDITED
  
  ---
  
  Sorry @username_2, I have 4 lanes for each samples (paired end) in each of experimental runs; For concatenating fastq files can I do like this ?
  
  ```
  cat fastq1_lane1_batch1 fastq1_lane1_batch2 fastq1_lane2_batch1   fastq1_lane2_batch2  fastq1_lane3_batch1 fastq1_lane3_batch2 fastq1_lane4_batch1 fastq1_lane4_batch2  > fastq1  
  
  cat fastq2_lane1_batch1 fastq2_lane1_batch2 fastq2_lane2_batch1   fastq2_lane2_batch2  fastq2_lane3_batch1 fastq2_lane3_batch2 fastq2_lane4_batch1 fastq2_lane4_batch2 > fastq2
  
  ```
  
  **Tags:** r, rna-seq, deseq2, pca, batch-effects
  
  > **Comments:**
  > **username_1**: Why do you want to collapse replicates? It's really good that you have replicates. Treat them as such.  
  > **username_2**: She's talking about technical replicates.  
  
  ---
  
  ## 2 answers
  
  ### Answer by username_2 on 2019-09-15. Score: 3 | Accepted answer
  
  Yes, you can safely concatenate the technical replicates. Odds are good that these are even the same libraries just sequenced twice, so even labeling them as replicates is a bit of a stretch. As an aside, it would be surprising if you actually had a batch effect in a situation like this. You will commonly see sequencing facilities just sequence a given sample a second time if it didn't get the number of reads requested by the client to begin with. They then just have the client concatenate the files.
  
  > **Comments:**
  > **username_0**: Sorry @username_2, I have 4 lanes for each samples (paired end) in each of experimental runs; For concatenating fastq files can I do like this ?                  cat          fastq1_lane1_batch1 fastq1_lane1_batch2 fastq1_lane2_batch1   fastq1_lane2_batch2  fastq1_lane3_batch1 fastq1_lane3_batch2 fastq1_lane4_batch1 fastq1_lane4_batch2                             fastq2_lane1_batch1 fastq2_lane1_batch2 fastq2_lane2_batch1   fastq2_lane2_batch2  fastq2_lane3_batch1 fastq2_lane3_batch2 fastq2_lane4_batch1 fastq2_lane4_batch2  
  > **username_3**: @Fereshteh That's a different question, and one you can find answers to by searching this site or biostars. Please do not stretch people offering to help by piling on more questions.  
  > **username_0**: Sorry @username_2 I think this question relates to this post; Can I use technical replicates for gene differential expression analysis? For example I am only interested in tumour A and normal A , can I compare them by using batch1 and batch2 as my replications?  
  > **username_2**: I think you know the answer to that is "no".  
  
  ---
  
  ### Answer by username_4 on 2019-09-16. Score: 1
  
  Prepping the RNA on different days, or making Illumina libraries on different days, or having different technicians handle different samples; that can lead to batch effects.
  Running samples on two different days does not cause a significant batch effect, as you can plainly see in your PCA. You should just combine the fastqs.
  
  > **Comments:**
  > **username_0**: Thank you @username_4, now my question is can I compare only one tumour with its own matched normal sample to get differentially expressed genes for this pair by using experimental runs (technical replicates )? Design would be Tumour1_batch1 Tumour1_batch2 versus Normal1_batch1 Normal1_bacth2  
  > **username_2**: @Fereshteh As you're well aware, new questions should be posted as such.  
  
  ---
  
  