Question: Knock out genes and TPM
gravatar for simang5c
8 weeks ago by
simang5c10 wrote:

why do deleted genes still show significant TPM? In some cases even higher than the wild type? I checked the alignment using IGV viewer but couldn't find much difference, any help would be much appreciated.

Thanks in advance!

ADD COMMENTlink written 8 weeks ago by simang5c10

Please explain more about the experiment to which you are referring. In bioinformatics, dark forces are always at play...

ADD REPLYlink written 8 weeks ago by Kevin Blighe69k

Sorry, I'll try sharing some more details.

We have 3 fungal strains from which we knocked out single-copy genes using Split-Marker Recombination. We then performed Transcriptomics sequencing and completed the bioinformatics analysis. We used STAR for aligning and RSEM for quantification. Following quantification, when I checked the TPM values of the deleted genes, some deleted genes showed less TPM(<1 TPM in the mutant), while some deleted genes showed higher TPM (30x times higher than Wild type) even after being deleted. Such a high TPM value of some deleted genes left me wondering if they were properly deleted or if there could be some bioinformatics issues that led to such results.

Any help regarding this topic would be much appreciated.

P.S. I also check the alignment results using IGV for those genes which showed high TPM values but it showed no much considerable difference when compared to the Wild type.

ADD REPLYlink written 8 weeks ago by simang5c10

It would be nice to know what you want to do precisely. If you want to test what genes are changing between 2 different conditions (the so called differential expression analysis), TPM are not the right way to go. I guess you want to see which genes are different between your strains?

ADD REPLYlink written 8 weeks ago by jordi.planells330

Thanks for the comment @jordi. Yes, we performed differential expression analysis as well. However, the main question that came up after observing the TPM values of deleted genes in mutant and Wild was, if the gene is knocked out, its TPM values should be 0 (ideal case) or less than 1 TPM in all samples except in wild type? Can high or low TPM values of knockout genes be correlated to whether the gene is deleted or not? For example, deleted genes should have TPM less than 1 or lower than the TPM value as seen wild type and so on?

ADD REPLYlink modified 8 weeks ago • written 8 weeks ago by simang5c10

How did you do the knockout? Did you used siRNAs? In that case, it is normal that you find that your KO gene is extremely high expressed (TPM). This is because the siRNAs (you normally transfect with high concentration) is captured by the library prep kit and sequenced. However if you look at the protein levels (WB), you shouldn't detect it. Does it make sense to you?

ADD REPLYlink written 8 weeks ago by jordi.planells330

Thanks for your input here jordi

ADD REPLYlink written 8 weeks ago by Kevin Blighe69k

No problem! It's my pleasure.

ADD REPLYlink written 8 weeks ago by jordi.planells330
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