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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

And this is biplot of samples enter image description here

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

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
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  • $\begingroup$ Why do you want to collapse replicates? It's really good that you have replicates. Treat them as such. $\endgroup$ – burger Sep 15 at 20:34
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    $\begingroup$ She's talking about technical replicates. $\endgroup$ – Devon Ryan Sep 15 at 21:36
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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.

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  • $\begingroup$ 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 $\endgroup$ – Angel Sep 15 at 23:40
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    $\begingroup$ @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. $\endgroup$ – Ram RS Sep 15 at 23:50
  • $\begingroup$ 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? $\endgroup$ – Angel Sep 16 at 16:35
  • $\begingroup$ I think you know the answer to that is "no". $\endgroup$ – Devon Ryan Sep 16 at 18:15
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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.

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  • $\begingroup$ 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 $\endgroup$ – Angel Sep 16 at 16:48
  • $\begingroup$ @Fereshteh As you're well aware, new questions should be posted as such. $\endgroup$ – Devon Ryan Sep 16 at 18:14

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