Question: Differential expression issue
0
gravatar for sabaghianamir70
7 months ago by
iran
sabaghianamir7010 wrote:

Hello

I having a problem with analyzing my data.. I have 5 time points of 5 patients, So the problem is, when i take all timepoints 0 from all patients and compare them with timepoints 1 from all patients, my p value adj failed. P.s i cant compare each person separately, because i just have one sample per each timepoint, So i at least need two replicate of each sample to do DEG.

Any suggestions are appropriated. Thanks.

rna-seq • 193 views
ADD COMMENTlink modified 7 months ago • written 7 months ago by sabaghianamir7010
1

i just have one sample per each timepoint

May I ask why the experiment was designed in this way? Which differential expression analysis program are you using?

ADD REPLYlink written 7 months ago by Kevin Blighe66k

Deseq and edgeR. i tried both. As i said : I thought i can compare all time 0 from 5 patients to all time 1.

ADD REPLYlink written 7 months ago by sabaghianamir7010

Thanks, but you are contradicting yourself with these statements:

I thought i can compare all time 0 from 5 patients to all time 1.

i just have one sample per each timepoint

I have 5 time points of 5 patients

Can you please clarify all samples that you have, and what is the specific issue? Is the issue just that no gene passes FDR correction? In that case, perhaps there genuinely is no statistically significant difference. You could try to reduce the 'FDR burden' by eliminating low count genes prior to normalisation, if not already done.

Explaining your experimental design and conditions may also further help us to understand any other potential issues. For example, you may need to adjust for confounding factors or other sources of bias.

ADD REPLYlink modified 7 months ago • written 7 months ago by Kevin Blighe66k

I have 5 patients which all have 5 timepoints.(0 1 3 5 7) So i just need to take all samples which belongs to time 0, and compare them to other time points For example : 0-1 0-3 0-5 0-7 The main issue is about my FDR and p value adj, because in when i run it with edgeR, i have no significant up or down. Also i have MDS plot like that which showing some thing is wrong.enter image description here My datas are Mir-seq and after cleaning i used bowtie and featureCount for further steps. I hope these information are what you want to know. Thanks a lot for helping :)

ADD REPLYlink written 7 months ago by sabaghianamir7010

Please show full code and how you set up the design matrix and contrasts.

ADD REPLYlink written 7 months ago by ATpoint40k

because I should pay for using cloud services, i just trying to test the whole thing in galaxy. If it helping, i can just send all workflow here.

Rscript '/opt/galaxy/shed_tools/toolshed.g2.bx.psu.edu/repos/iuc/edger/334ce9b1bac5/edger/edger.R'  -R '/data/dnb02/galaxy_db/job_working_directory/007/029/7029289/galaxy_dataset_14796253.dat' -o '/data/dnb02/galaxy_db/job_working_directory/007/029/7029289/working/dataset_13572063_files'                            -j '[["timecourse", [{"t14": ["/data/dnb02/galaxy_db/files/013/558/dataset_13558766.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558769.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558772.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558775.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558778.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558781.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558784.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558787.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558790.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558793.dat"]}, {"t0": ["/data/dnb02/galaxy_db/files/013/558/dataset_13558729.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558732.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558735.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558738.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558741.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558744.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558747.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558750.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558753.dat", "/data/dnb02/galaxy_db/files/013/558/dataset_13558756.dat"]}]]]'    -C 't14-t0'    -l '0.0' -p '0.05' -d 'BH' -n 'TMM' -b  && mkdir ./output_dir  && cp '/data/dnb02/galaxy_db/job_working_directory/007/029/7029289/working/dataset_13572063_files'/*.tsv output_dir/
ADD REPLYlink modified 7 months ago by RamRS30k • written 7 months ago by sabaghianamir7010

Thanks, but just the design matrix, please.

ADD REPLYlink written 7 months ago by russhh5.5k

enter image description here

this is how i set up my analysis..each Count file includes 5 time points.

ADD REPLYlink written 7 months ago by sabaghianamir7010

Ok, we are at a dead-end here. The problem that me and others were asking about is how the tool knows which samples belong to one group. This seems to be a blackbox since you are using Galaxy. Therefore, you have two options. First, see if you can get the raw count matrix and then do analysis locally, 100% following edgeR manual. Second, set Output Rscript to TRUE and then upload it somewhere, then please share the link. Then we can inspect what Galaxy is doing.

ADD REPLYlink modified 7 months ago • written 7 months ago by ATpoint40k

Yeah, well this does not help at all. If you cannot show code it is difficult/impossible to debug. Things to check are if experimental groups are properly set, if design matrix is correct and if the contrasts are properly set up, and how multiple testing correction was done. Get raw data and follow the edgeR manual, it covers all you need for standard analysis.

ADD REPLYlink written 7 months ago by ATpoint40k

Could you be more specific about why p-value adjustment "failed"? Is it just that you didn't observe any significant changes.

ADD REPLYlink written 7 months ago by russhh5.5k

enter image description here

As you can see in this picture, All p value adj are above the 0.05

ADD REPLYlink written 7 months ago by sabaghianamir7010

Ah. So they haven't failed, they're just not significant. Did you control for the different baseline expression level in your 5 patients?

ADD REPLYlink written 7 months ago by russhh5.5k

How can i check that ? I almost tried every option in edgR or Deseq2. can you explain how can i control the different baseline expression level ?

ADD REPLYlink written 7 months ago by sabaghianamir7010
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