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Creating a panel of normal from one normal sample

Hi,

I have just one normal sample and 3 tumors from the same patient. In this case is it ok to generate PoN using the following code?

gatk --java-options "-Xmx16g" Mutect2 \
    -R $ref \
    -I $BAMs/normal.bam \
    -tumor normal \
    --germline-resource somatic-hg38-af-only-gnomad.hg38.vcf \
    -O $out/normal_for_pon.vcf.gz

Can you use the normal_for_pon.vcf.gz for subsequent steps?

I couldn't find much information about using just one normal sample in the tutorials.

Best Answers

Answers

  • anishanish Member
    edited November 2019

    Hi using the above shared information I tried the following steps.

    gatk --java-options "-Xmx16g" Mutect2 \
        -R $ref \
        -I $BAMs/normal.recal.bam \
        --max-mnp-distance 0 \
        -O $out/normal.vcf.gz
    
    gatk GenomicsDBImport \
        --genomicsdb-workspace-path $out/pon_db \
        -R $ref \
        -L $interval_list \
        -V $out/normal.vcf.gz 
    
    gatk CreateSomaticPanelOfNormals \
        -R $ref \
        --germline-resource $g_resource \
        -V gendb://$out/pon_db \
        -O $out/pon.vcf.gz
    

    While steps 1&2, seemed to work. CreateSomaticPanelofNormals created an empty vcf file with just the header. And I also got the below warning:

    06:30:53.943 INFO  CreateSomaticPanelOfNormals - Initializing engine
    06:30:54.280 INFO  FeatureManager - Using codec VCFCodec to read file file:///zfs1/uchandran/Anish/Refs/GATK_Bundle/somatic-hg38-af-only-gnomad.hg38.vcf
    WARNING: No valid combination operation found for INFO field CONTQ - the field will NOT be part of INFO fields in the generated VCF records
    WARNING: No valid combination operation found for INFO field ECNT - the field will NOT be part of INFO fields in the generated VCF records
    WARNING: No valid combination operation found for INFO field GERMQ - the field will NOT be part of INFO fields in the generated VCF records
    WARNING: No valid combination operation found for INFO field MBQ - the field will NOT be part of INFO fields in the generated VCF records
    WARNING: No valid combination operation found for INFO field MFRL - the field will NOT be part of INFO fields in the generated VCF records
    WARNING: No valid combination operation found for INFO field MMQ - the field will NOT be part of INFO fields in the generated VCF records
    WARNING: No valid combination operation found for INFO field MPOS - the field will NOT be part of INFO fields in the generated VCF records
    WARNING: No valid combination operation found for INFO field NALOD - the field will NOT be part of INFO fields in the generated VCF records
    WARNING: No valid combination operation found for INFO field NCount - the field will NOT be part of INFO fields in the generated VCF records
    
  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    Hi @anish

    How many samples are you using to create your PON?

  • anishanish Member
    edited November 2019

    Just one normal, like I mentioned in my question.

  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    @anish

    We recommend using at least 40 samples to create a PON. Take a look at this doc: https://software.broadinstitute.org/gatk/documentation/article?id=11053

  • anishanish Member

    So, does it mean one cannot use one normal to generate PoN?

    There is no definitive rule for how many samples should be used to make a PON (even a small PON is better than no PON)

    Based on the above statement I assumed, though recommended is 40, having one is better than having none.

    Do you not recommend to use PoN if there are less than 40 normals?

  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin
    edited November 2019

    @anish

    There is an argument in CreateSomaticPanelOfNormals that you can change--min-sample-count. We do not recommend this. However, that is probably why you are getting an empty file. It is set by default to 2.

  • bhanuGandhambhanuGandham Cambridge MAMember, Administrator, Broadie, Moderator admin

    That is exactly right. Thank you @manolis ! We are grateful to you for always providing such valuable input!

  • anishanish Member

    Thank you both. I have a better understanding of things now!

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