Holiday Notice:
The Frontline Support team will be offline February 18 for President's Day but will be back February 19th. Thank you for your patience as we get to all of your questions!

Mutect2 AF calculation discrepancies


I called some targeted DNA-seq using GATK 4.0 mutect2 :

java -jar $gatk_jar Mutect2 \
     -R $ref_fasta \
     -I $bam \
     -tumor $sample \
     -L $genomic_interval_bed \
     --dbsnp $dbSNP_vcf \
     -O $sample"_mutect2.vcf.gz"

Works well but I've a question regarding the generated vcf. Looking at it I found some variants with calculated AF that do not reflect the number of alternate reads vs reference reads.

Example :

chr10 43597975 . C T . . DP=395;ECNT=1;POP_AF=1.000e-03;P_GERMLINE=-3.512e-04;TLOD=6.21 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB 0/1:313,6:0.112:125,6:188,0:30:80,90:60:15:0.010,0.020,0.019:0.011,1.407e-03,0.988

If I understood this line, I've 395 reads in total. 313 reference and 6 alternate looking at the AD tag. AF=0.112

  • First question : 313+66 != 395. Did I misinterpret the DP and AD tag ?

  • Second question : 6/395 != 0.112 . How is the AF tag computed ?

    Did I miss something here ?

Thank you


  • NicoBxlNicoBxl Member
    edited March 2018

    I dig a little bit and plotted the Mutect2 computed AF versus a classic Alt/ (Alt+Ref). Mutect2 AF is always higher than the standard Alt/(Alt+Ref) for low AF variants (see boxplot) . So which AF to use for further filtering as some variants can have Mutect2 AF ~= 0.1 and Alt/(Alt+Ref) AF ~= 0.01 ..

    Thank you for the clarifications.

  • SheilaSheila Broad InstituteMember, Broadie, Moderator admin


    1) Have a look at this article.

    2) Have a look at this thread, specifically my response at the end.


Sign In or Register to comment.