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Variant Discovery in High-Throughput Sequencing Data

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Developed in the Data Sciences Platform at the Broad Institute, the toolkit offers a wide variety of tools with a primary focus on variant discovery and genotyping. Its powerful processing engine and high-performance computing features make it capable of taking on projects of any size. Learn more

Mutect2 for ultrasensitive low coverage calling



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    Genevieve Brandt (she/her)

    We have this documentation, troubleshooting how to make Mutect2 and HaplotypeCaller more sensitive. For low coverage, we recommend to run the analysis with  --min-pruning 0.

    There are many ways you can run Mutect2, examples are in the documentation. Please post your entire command and GATK version number so that others can see what you have tried, and what they might recommend you change. 

    The GATK support team is focused on resolving questions about errors and abnormal results, so for questions regarding best parameters like this, we ask the community to chime in with what has worked for them. 

    Please continue to post your questions because we will be mining them for improvements to documentation, resources, and tools.

    For context, check out our support policy.

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    David Benjamin

    I would try running Mutect2 in multi-sample tumor-normal mode if I were you:


    gatk Mutect2 -R ref.fasta -I tumor1.bam -I tumor2.bam . . . -I tumor50.bam \

       -I normal.bam -normal normal_sample_name_in_bam_header \

       -germline-resource af-only-gnomad.vcf.gz \

       -pon pon.vcf \

       -O unfiltered.vcf


    gatk FilterMutectCalls -R ref.fasta -V unfiltered.vcf -O filtered.vcf


    The knobs to adjust to increase sensitivity in Mutect2 (at the expense of runtime) are -pruning-lod-threshold and -tumor-lod-to-emit.  At very low values these may actually harm sensitivity as the assembly graph will get clogged with errors.

    To increase sensitivity in FilterMutectCalls at the expense of precision you may adjust the -f-score-beta argument.

    -callable-depth, by the way, sets the threshold for counting callable sites in the .vcf.stats output in order to help FilterMutectCalls learn the prior probability of somatic variation.  It is not a hard threshold for variant calling and we do not recommend adjusting it.

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