Test-drive the GATK tools and Best Practices pipelines on Terra
Check out this blog post to learn how you can get started with GATK and try out the pipelines in preconfigured workspaces (with a user-friendly interface!) without having to install anything.
MuTect2 all potential somatic mutations did not pass the alt_allele_in_normal filter
I am trying to use MuTect2 to call low-frequency (0.1-5%) mutations in yeast population but none of them passed the alt_allele_in_normal filter, meaning the allele is presented in control ("normal") sample. However, it is not look reasonable as soon as I used control to create Panel of Normals and my samples successfully passed the panel_of_normals filter. In addition, when I visualized the reads through the IGV, I did not see any alternative alleles in control at these positions.
Briefly, my experiment design: I poolled 1000 yeast colonies (after certain mutagenic conditions), extracted DNA and amplified a gene of interest through the PCR. Then I purified the amplicons and sequenced with MiSeq Illumina platform, paired-end. The final coverage is between 10 000 - 30 000 (10-30 per genome). I preproceced the data by GATK best practices and I created PON trough the artifact detection mode based on the same protocol with wild type strain. At least one mutation is expected per colony.
Finally, I ran Mutect2 with following parameters:
GenomeAnalysisTK -T MuTect2 -R path_to_reference/reference.fa -I:tumor path_to_file/realigned_Sample1.bam -I:normal path_to_file/realigned_control.bam -L I:X1-X2 --sample_ploidy 1000 -PON output.control.vcf -o output_control_vs_sample1.vcf
Could you help me to fix this problem?