Variants Calling on RNA Seq Samples

Question

[benjyrolls]

hello

I have 4 RNA Seq samples from p53 wild type mouse(WT) and p53 knock out(KO) mouse (2 replicates each) which were treated with Tamoxifen .

these were analysed with sam tools to call variants. Variants were called for each of the WT and KO samples.(4 .vcf files)

my question is even though variants were found , is it genomically important to call variants on each of these 4 samples and are they biologically meaningful ?

Are variants only called on Tumour and Normal tissues or they can be called on an RNA seq transcription factor KO experiment?

Thanks

benjy

[WouterDeCoster]

is it genomically important to call variants on each of these 4 samples and are they biologically meaningful ?

Are variants only called on Tumour and Normal tissues or they can be called on an RNA seq transcription factor KO experiment?

What is the biological question you are trying to answer?

[benjyrolls]

i am trying to figure the effect of the p53 knockout on the deferentially expressed genes and whether the deferentially expressed genes are linked to a specific biological process. However, in my RNA Seq pipeline there is an option to check for variants, i wanted to understand the meaning of those variants called.

[WouterDeCoster]

So, assuming you are using inbred mice the mice are identical except for the knockout of p53, right? Then what would be the added value of investigating mutations?

[benjyrolls]

ok, does this mean on the mutations from the p53 knockout are significant in this case since the Wild Type mice could be identical ? Will it be worth considering the mutaions from the two KO replicates in terms of the effect of p53 KO ? i have attached a sample output from the .vcf from one of the Knock out mouse https://ibb.co/iOnfxa

[Devon Ryan]

Your KO mice are also genetically identical, or at least they should be. Hopefully your WT line is the same genetic background, so there is then absolutely no benefit to doing variant calling. This is also what any biologist in the world would have told you.

[ivivek_ngs]

It is a classic example when a newbie Bioinformatics person is left in a dark room with a PC to find the light that leads to the exit path. Pretty much makes me think both the Biologist and the Bioinformaticians have the least idea of what they want to do and what are the aims of the experimental system. Tbh, this issue should be taken care by the mentor when such situation arises else both time and computing resources will be wasted. I am just amazed that a variant calling was performed for genetically identical mice where the system has a WT and a KO. Please understand the design of the experiments and why these datasets were generated. If you cannot buzz your mentor about the project plan and why such design was made then try to self-study from similar experimental designs that have been published. It is not about finding a needle in the haystack. There is a certain hypothesis already derived on expression datasets of WT and KO p53 mice. Read those papers and reviews and see how these fit well with your lab projects. This will be a way for your to understand the need of such experimental designs.