My research is focused on Wilms tumor1 gene mutations in acute myeloid leukemia and we use bone marrow or peripheral blood samples; therefore, it is almost unlikely that I could be able to find a pure tumor samples and there is always a background of normal (wild types cells) present. So, in this context, what is the best method to figure out if the Wt1 mutations in our samples are monoallelic or biallelic?
That is a good question. Another complicating factor is the fact that the second allele might be deleted also, which has the same affect as a deleterious mutation on both alleles. Moreover, there might be substantial subclonality/heterogeneity. This makes the question even harder... Effectively, you will end up with an educated guess
In a first step you could try to quantify the contaminating normal fraction. If you analyze more than the single WT1 mutation you should come up with several somatic mutations (regardless whether they are driver or passenger mutations) and come up with mutant allele frequencies, where most should be monoallelic. If the mean/median of those is around 40% you should have somewhere around 80% tumor fraction. If your mutant allele frequency (i.e. the reads supporting the mutation vs. the reads supporting the wild-type allele) of the WT1 mutation is around 80% it should be biallelic, 40% > monoallelic. However, beware that there are uncertainties and assumptions, which might be wrong all over the place and a deletion of the second allele might screw all your numbers.
If you also happen to analyze copy-number alterations across the genome, you could try to calculate the tumor fraction of that using e.g. ABSOLUTE (http://archive.broadinstitute.org/cancer/cga/absolute). This way you would also be able to see a possible deletion of WT1.
Still with all these, you will not end up with definitive conclusions...
Hope that helps,
Best
Peter
if there was a snp nearby you could ARMS amplify a product from the allele of the snp that was in linkage disequilibrium with the mutation you are interested in.. Failing that pyrosequencing is very good for accurate measurement of an allele in the presence of another allele
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