Detection of mutations in plasma samples using multiplex amplicon sequencing - (Apr/24/2014 )
I am trying looking for genetic mutations in plasma. This was done by multiplex amplicon sequencing using custom primers. Variant calling was done with a tool in the SamTools suite, called mPileup. Two filters based on the variant allele frequency (more than 0.5%) and ratio of variant to 3rd highest base ( at least 15), were used to remove low quality reads.
I am detecting very few variant reads in our plasma samples.
Does anyone have an idea why this happens? What could be a cause of this result? I am hoping to get more variants in my plasma sample
What approach are you using to normalize your samples (spec, quantitative binding, etc...). You could go to UCSC and find a common SNP and compare the reported frequency to your sample. Does your raw data indicate that you have good quality reads?
I'm not familiar with mpileup, but in our suite that we use you can adjust how each base is called to increase the number of your variants - again you can use common SNP frequencies to adjust the variant calling.