If you see most of your relevant mutations G>A, what would you infer (generally) other than any sequencing error. Chances of sequencing errors are already addressed. Data generated on Prostate Cancer Tissue sample. All mutations are somatic mutations and most of the mutations are validated (greater than 100 X depth in primary sequence data)
increased cytosine deamination (to form uracil which will pair with A) in your tumors maybe?
Hi,
You should read the paper in attachment, I think it will give you some clues about biologic hypotheses behind it.
best,
Ludovic
Article Signatures of mutational processes in human cancer
As well as the mentioned biological changes in the source DNA, there are also chemical changes to the DNA during PCR, e.g. depurination and deamination. Both are pH dependent, so buffer composition will affect the perceived error rate. Try to limit the time and temperature of the denaturation step to minimise such damage.
For more explanation on these artifactual processes, start with
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1544370/
and follow references/links/terminology searches.
Good luck!
My bet would be also on Cytosine Deamination and the inability to discern G>A from C>T mutations on the other strand. However, I would blame fixation. Is your sample formalin fixed? We do see that a lot and neither the somatic status (I guess the normal DNA was not paraffin fixed?) nor the high coverage can get rid of that...
Increasing the sequencing depth will result in finding more somatic mutations (PCR and sequencing artefacts and real somatic mutations), because they will come above the treshold settings and become vissible in more then one read. G>A and C>T are mutation hotspots as explained by others already.
Thanks for all your answers...Peter: yes those tissues were formalin fixed....
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