Question: DESeq2 with several biological replicates
0
gravatar for bharata1803
5 months ago by
bharata180340
Japan
bharata180340 wrote:

Hello,

Is there any guide or tutorial to process RNA-seq sample with biological replicates? I have 2 conditions, normal and tumor. The total number of sample is 20 with each normaland tumor has 5 biological replicates. Thanks.

ADD COMMENTlink modified 5 months ago • written 5 months ago by bharata180340
Answer: DESeq2 with several biological replicates
0
gravatar for Michael Love
5 months ago by
Michael Love22k
United States
Michael Love22k wrote:

Can you describe these biological replicates a bit more? How many patients are there here? Are the tumors and normals matched?

ADD COMMENTlink written 5 months ago by Michael Love22k

The number of patients is 20. Each patients has 5 normal rna-seq reads and 5 tumor rna-seq reads (matched).

ADD REPLYlink written 5 months ago by bharata180340

Let me rephrase. You have patient 1, normal and tumor. You have 5 files for patient 1 normal. Are these from different parts of the adjacent tissue? Are they from different day? Are they the same library sequenced 5 times? Likewise, you have 5 files for patient 1 tumor. Are these from different parts of the tumor? Are they from different day? Are they the same library sequenced 5 times? What was the point of the 5 libraries rather than 1 library?

ADD REPLYlink written 5 months ago by Michael Love22k

Actually it is a public data. So, I am not so sure myself. This is the link: https://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP069212

From the paper, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5316832/ they only mentioned like this:

Total RNA from 60 samples from 20 Chinese HCC patients was sequenced (GSE77509). Each patient had three matched samples: primary HCC tumour, adjacent normal liver tissue and PVTT. The patients were ordered using alphabetic labels (A to T) in this paper, but the patients were originally numbered as 3,6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25 and 26. The PVTT sample of one patient (14) was not distinguishable from normal tissue, so we did not use it in our migration and metastasis analyses. The ethical committee of EHBH hospital approved this study. Informed consent was obtained from each patient.

I have used the DESeq2 to get DE genes from their normalized readcount data. Now I want to process the data from the beginning.

From what I understand, I think it is the same sample sequenced 5 times.

ADD REPLYlink modified 5 months ago • written 5 months ago by bharata180340

Without more information on what the samples mean, I can’t provide any advice on how to treat them.

ADD REPLYlink written 5 months ago by Michael Love22k

hello, it seems it is not biological replicates but technical replicates. the biological sample is the same. it just run/sequenced 5 times. Can you give some suggestion? If not, I will just take 1 sample from every patient to make things simpler. If it is from the same sample and just sequence 5 times, it won't make any difference in the biological sense.

ADD REPLYlink written 5 months ago by bharata180340

We have code in the vignette for collapsing technical replicates. Take a look there.

ADD REPLYlink written 5 months ago by Michael Love22k
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