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Question: DESeq2 with several biological replicates
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gravatar for bharata1803
12 days ago by
bharata180320
Japan
bharata180320 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 12 days ago • written 12 days ago by bharata180320
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gravatar for Michael Love
12 days ago by
Michael Love20k
United States
Michael Love20k 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 12 days ago by Michael Love20k

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

ADD REPLYlink written 12 days ago by bharata180320

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 12 days ago by Michael Love20k

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 11 days ago • written 11 days ago by bharata180320

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

ADD REPLYlink written 11 days ago by Michael Love20k

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 8 days ago by bharata180320

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

ADD REPLYlink written 7 days ago by Michael Love20k
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