MuTect2 all potential somatic mutations did not pass the alt_allele_in_normal filter

Hello,

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?
Thank you!

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