Hi
I have RNA-seq from two batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2. He says that one batch shows under counting and another one shows over counting. Now I have extracted two independent matrices of raw read counts from batches 1 and 2 but I don't know if I must run DESeq2 on each batch independently or I can take mean of read counts over two batches? Actually I am not sure what people do in this situation I just know he says everything has been the same in running experiments but he has run the RNA-seq two times
Thank you for any help
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zizigolu
Hey,
In your metadata, create a new variable called batch, and introduce this as a covariate in your DESeq2 design formula.
For example:
ID batch condition
sample1 first case
sample2 first control
sample3 second case
sample4 first control
sample5 second case
sample6 second control
Then, the design formula:
~ condition + batch
This will have the effect of adjusting the test statistics for batch.
Take a look at this very interesting thread: https://support.bioconductor.org/p/76099/#76101
Kevin
Sorry I have same samples for both batches 1 and batch 2 ; I mean experiment has been run two times on the same patients
For example tumor 1 in batch 1 and tumor 1 in batch 2
normal 2 in batch1 and normal 2 in batch 2
Does this make any difference in your suggested design?
If they are from the same biopsy and library preparation, then just concatenate the FASTQ files together.
If it is tumour DNA, then 2 samples from the same biopsy can still be very different (tumour heterogeneity). Ultimately, processing them both separately and then combined will reveal more information to you.
@Kevin
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
>
By your package I got this bioplot by vsd values
![enter image description here][1]
From biplot, batch1 and batch2 of each patient are close to each other, Can I then collapse technical reads to each other?
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