mice whole Exome sequencing Analysis

Question

[tarekzakaria.badr]

Hi everyone,

i am a little bit new to whole exome seq analysis and did it only once before for human samples using bwa and Gatk pipeline and now I want to do whole exome sequencing of "tumor biopsies" from 12 mice (6 Ko and 6 ctrl). The main goal is to find difference in number and nature/patterns of mutations between the tumor samples from the ctl vs ko mice ( as non synonymous, synonymous, non coding, frequency of transitions, etc..)
as we believe the ko to be more protected (therefore should accumulate less number of mutation).

I would like to know your suggested depth of coverage (i initially thought about 100x at least), and a suggested analysis workflow would be much appreciated.

Thanks in advance.

[ATpoint]

The goal of your endeavour would be helpful.

[tarekzakaria.badr]

Hi, just updated the post and added some details to it :) i am actually planning to sequence tumor biopsies from the two mice genotypes to see how the the Knockout would affect the tumor nature and frequency of mutation accumulation to see how they would affect the tumor aggressiveness and prognosis as we believe the knockout to be more protected and should have more benign/ less aggressive tumor and therefore less number of harmful mutations. Thanks in advance.

[ATpoint]

I of course have no insight into your research, but wouldn't a knockout that has beneficial effects suggest that you should rather check for transcriptional changes and/or epigenetic effects such as histone modification patterns and open chromatin landscape on regulatory elements?

[tarekzakaria.badr]

Thanks for your feedback and Sorry for the late reply. I believe RNAseq would be conducted be our collaborators too and basically they would like to see the difference in mutation load between the two mice strains (i believe they are inspired with this paper https://www.sciencedirect.com/science/article/pii/S153561081730507X?via%3Dihub).

[h.mon]

Is it whole genome or exome? Your title says genome.

Unless you want to perform diagnostics on these mice, I don't think you need >100x coverage, probably around 50x will suffice. Are you trying to detect off-site mutations?

[tarekzakaria.badr]

Hi, it's exome whole seq. sorry for the typo, was writing from my mobile :) just updated the post and added some details to it as my initial post wasn't that clear. i am actually planning to sequence tumor biopsies from the two mice genotypes to see how the the Knockout would affect the tumor nature and frequency of mutation accumulation to see how they would affect the tumor aggressiveness and prognosis. Thanks in advance.

[shubhra.bhattacharya]

You can look into mutect: https://software.broadinstitute.org/gatk/documentation/tooldocs/3.8-0/org_broadinstitute_gatk_tools_walkers_cancer_m2_MuTect2.php

[tarekzakaria.badr]

Thanks a lot for your feedback and sorry for the late reply. That's an interesting pipeline. i used GATK before for normal human exome and found it convenient. A small question regarding the pipeline (sorry if it seems naive but i am really new to tumor research). We have tumor samples from the two mice strains with no normal tissue from these mice (i believe the mice are already sacrificed). is it possible to call the variants in the tumor tissue from the two strains without the normal tissue and compare the mutation load between them?