about Mutect2 usage

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Sukjung Choi

• March 04, 2020 07:43

a) gatk-4.1.0.0 version

b) java 1.8

c) commands used

/usr/local/lib/gatk-4.1.0.0/gatk --java-options "-Xmx2g" Mutect2 \
-R /data/Reference/GRCh38/GRCh38.p12.genome.fa \
-I tumor.bam \
-I normal.bam \
-tumor tumor.bam \
-normal normal.bam \
-O 1_somatic_m2.vcf.gz \
-bamout 2_tumor_normal_m2.bam

d) Actually, after running Mutect2, I received "success" message

16:35:02.441 INFO Mutect2 - Shutting down engine
[March 4, 2020 4:35:02 PM KST] org.broadinstitute.hellbender.tools.walkers.mutect.Mutect2 done. Elapsed time: 20.71 minutes.
Runtime.totalMemory()=1856503808
Tool returned:
SUCCESS

However, vcf file does not contain varients like below

##filtering_status=Warning: unfiltered Mutect 2 calls. Please run FilterMutectCalls to remove false positives.
##normal_sample=10001625-TN.replaced.bam
##source=Mutect2
##tumor_sample=10001625-TT.replaced.bam
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT 10001625-TN.replaced.bam 10001625-TT.replaced.bam

========================================================

I tested another bam files, but none of the varients were detected

Any problem on my commands?

 

And, I changed the bam header like below to add read group(@RG)

java -jar picard.jar AddOrReplaceReadGroups \
I=normal.bam \
O=normal.bam \
ID=10001625-TN LB=10001625-TN-1 PL=illumina PU=10001625-TN SM=bam file full name

Regards

0

• 

  David Benjamin

  • March 05, 2020 19:31

  Sukjung Choi I'm confused because the Mutect2 command says that the normal sample name is normal.bam, but the output VCF header says it is 10001625-TN.replaced.bam.  Could you double-check that the command line you posted is exactly the command you used to generate this output?

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  Sukjung Choi

  • March 06, 2020 05:12

  Thank you for your comment, David

  my command is like that

  /usr/local/lib/gatk-4.1.0.0/gatk --java-options "-Xmx2g" Mutect2 \
  -R /data/Reference/GRCh38/GRCh38.p12.genome.fa \
  -I 10001625-TT.replaced.bam \
  -I 10001625-TN.replaced.bam \
  -tumor 10001625-TT.replaced.bam \
  -normal 10001625-TN.replaced.bam \
  -O 1_somatic_m2.vcf.gz \
  -bamout 2_tumor_normal_m2.bam

  So, I add disable option

  --disable-read-filter MappingQualityAvailableReadFilter \

  Then, I could detect varients.

  Is it all right?

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  David Benjamin

  • March 06, 2020 16:37

  If disabling the mapping quality available read filter is necessary to call variants, then something is wrong with your bam file.  Because mapping artifacts are so common, it is very important for Mutect2 to have access to mapping qualities.  What aligner are you using?  Is there anything in your pipeline that might strip mapping qualities off of reads?

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