<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>

CC BY-SA 4.0
2019-09-15T23:40:28.323Z
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
Zizogolu
CC BY-SA 4.0
2019-09-15T23:50:54.503Z
@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.
Ram RS
CC BY-SA 4.0
2019-09-16T16:35:59.937Z
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?
Zizogolu
CC BY-SA 4.0
2019-09-16T18:15:11.400Z
I think you know the answer to that is "no".
Devon Ryan
CC BY-SA 4.0
2019-09-15T21:39:02.377Z
2019-09-15T21:39:02.377Z
Devon Ryan
<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>

CC BY-SA 4.0
2019-09-16T16:48:55.960Z
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
Zizogolu
CC BY-SA 4.0
2019-09-16T18:14:27.960Z
@Fereshteh As you're well aware, new questions should be posted as such.
Devon Ryan
CC BY-SA 4.0
2019-09-16T16:39:21.463Z
2019-09-16T16:39:21.463Z
swbarnes2
<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>

CC BY-SA 4.0
2019-09-15T20:34:34.793Z
Why do you want to collapse replicates? It's really good that you have replicates. Treat them as such.
burger
CC BY-SA 4.0
2019-09-15T21:36:19.930Z
She's talking about technical replicates.
Devon Ryan
<r><rna-seq><deseq2><pca><batch-effects>
Interpreting this PCA plot for RNA-seq
# 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

And this is biplot of samples

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 

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 @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  > 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 @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  
> **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 @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?  
> **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 @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  
> **username_2**: @Fereshteh As you're well aware, new questions should be posted as such.  

---

