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zhaopeihua 10-23-2013 03:54 AM

variant allele fraction
 
hi:
What are "variant allele fraction" when talking about somatic mutations?

lethalfang 10-23-2013 03:53 PM

Quote:

Originally Posted by zhaopeihua (Post 119687)
hi:
What are "variant allele fraction" when talking about somatic mutations?

The fraction of alleles in the sample that are mutations.
For instance, if all cells contains the same heterogeneous mutation (e.g., 100% tumor cell purity if you're looking at tumor cells), the variant allele fraction would be 50% (half-half from each chromosome).

zhaopeihua 10-23-2013 11:13 PM

Quote:

Originally Posted by lethalfang (Post 119746)
The fraction of alleles in the sample that are mutations.
For instance, if all cells contains the same heterogeneous mutation (e.g., 100% tumor cell purity if you're looking at tumor cells), the variant allele fraction would be 50% (half-half from each chromosome).

thanks for you reply,but I didn't fully understand this concept. Could you show me a detail example with computational process? thanks again

lethalfang 10-23-2013 11:27 PM

This is an example of an output in my somatic calling pipeline:

Code:


Variant Annotation        Gene        Exon        Codon        Chr        Position        Variant        N(A)        N(C)        N(G)        N(T)        T(A)        T(C)        T(G)        T(T)        QSS        Frequency
nonsynonymous SNV        KRAS        exon2        G12A        chr12        25398284        G35C        0        83        0        0        1        37        12        0        57        0.24


N(A), N(C), N(G), and N(T) stand for the number of calls for A, C, G, and T in the normal sample. T(A), T(C), T(G), and T(T) stand for the number of calls for A, C, G, and T in the tumor sample.

So for the normal sample, there are 83 sequences where the base is called C, and 0 for everything else. So it's pretty clear that in the reference and the normal sample, the base is C.
However, in the tumor sample, there are 37 calls for C (reference) and 12 calls for G (variant), so the variant frequency is 12/(12+37) = 0.245.
Notice the variant is labeled G35C. That's because the coding strand is the minus strand, where the DNA reads use the plus strand.

Anyway, how do you interpret 24.5%?
Well, if I assume that
1) the KRAS mutation is present in all tumor cells (just use this assumption as an example), and
2) KRAS mutation is a heterozygous mutation, then
The tumor sample must contain 1/2 non-tumor cells, because those cells give me half of the reference reads. Out of the remaining 1/2 are tumor cells, half of those chromosomes give me reference reads, and the other 1/2 chromosomes give me mutant reads.

zhaopeihua 10-24-2013 01:12 AM

Awesome, thanks!

vd4mindia 09-03-2014 05:18 AM

@lethalfang
`Would you like to share the pipeline for finding somatic mutations. I have 2 samples, one low and one high grade tumor and its normal. I want to detect the point somatic mutations. I have already employed GATK, VarScan and Mutect, but I have some descrepancy when am looking at my Low grade tumor since it is 50% pure. So I would like to try something more and have a read statistics like you have shown to validate the hits. If its ok for you

lethalfang 09-10-2014 10:12 AM

Quote:

Originally Posted by vd4mindia (Post 149028)
@lethalfang
`Would you like to share the pipeline for finding somatic mutations. I have 2 samples, one low and one high grade tumor and its normal. I want to detect the point somatic mutations. I have already employed GATK, VarScan and Mutect, but I have some descrepancy when am looking at my Low grade tumor since it is 50% pure. So I would like to try something more and have a read statistics like you have shown to validate the hits. If its ok for you

That's something I used to do a couple of years ago.
I just had a script to get those information out of the Strelka output vcf file. I had all those Strelka out directories in a single location, and ran this script to "summarize" all mutation calls from all the samples:

http://kimlab.surgery.ucsf.edu/media...a_findings.txt

It also calls annovar, which has a hard-coded path within the script.

virpericulosus 03-04-2015 05:30 AM

Just wondering: The T(A) isn't counted because the frequency is just for T(G) variants?

vd4mindia 03-25-2015 11:43 AM

Quote:

Originally Posted by lethalfang (Post 119775)
This is an example of an output in my somatic calling pipeline:

Code:


Variant Annotation        Gene        Exon        Codon        Chr        Position        Variant        N(A)        N(C)        N(G)        N(T)        T(A)        T(C)        T(G)        T(T)        QSS        Frequency
nonsynonymous SNV        KRAS        exon2        G12A        chr12        25398284        G35C        0        83        0        0        1        37        12        0        57        0.24


N(A), N(C), N(G), and N(T) stand for the number of calls for A, C, G, and T in the normal sample. T(A), T(C), T(G), and T(T) stand for the number of calls for A, C, G, and T in the tumor sample.

So for the normal sample, there are 83 sequences where the base is called C, and 0 for everything else. So it's pretty clear that in the reference and the normal sample, the base is C.
However, in the tumor sample, there are 37 calls for C (reference) and 12 calls for G (variant), so the variant frequency is 12/(12+37) = 0.245.
Notice the variant is labeled G35C. That's because the coding strand is the minus strand, where the DNA reads use the plus strand.

I would just want to ask you in case you have have not other bases as 0 in normal in that case do you still calculate the variant allele frequency as the above? Variant allele frequency should be always considered for the reads having variant allele for the tumor sample divided by the total reads at that variant site? Right? Irrespective of the reads falling for other bases in normal samples as well right?

lethalfang 03-25-2015 11:46 AM

Quote:

Originally Posted by vd4mindia (Post 163136)
I would just want to ask you in case you have have not other bases as 0 in normal in that case do you still calculate the variant allele frequency as the above? Variant allele frequency should be always considered for the reads having variant allele for the tumor sample divided by the total reads at that variant site? Right? Irrespective of the reads falling for other bases in normal samples as well right?

I considered "variant allele frequency" as the frequency of variant alleles in a given sample.
So the tumor's variant allele frequency does not care what's in the normal tissue, and vice versa.
That way, you can monitor the change in variant allele frequency between the tumor and the normal.

vd4mindia 03-26-2015 08:01 AM

Thank you very much for the reply. That makes sense.


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