Hello,
we are working with Mutect2 to call somatic variants (snvs and indels) on targeted sequencing. We recently have noticed an issue related to an specific variant detection, so that we detect it if we have an small interval within is the variant, but if this interval is greater, it is not detected.
These are both intervals:
chr14 104780203 104780225
chr14 104780078 104780226
The interest variant is in the chr14 104780214 genomic position (within both intervals), and we are able to see it using IGV with an AF of 66%, as it is shown in the image (sample_bam_igv.png).
The command we are using is the following (we also run this with last gatk version, gatk-4.0.11.0, and have the same result):
gatk-4.0.5.0 --java-options "-Xmx30g" Mutect2 --native-pair-hmm-threads 20 -R ~/Homo_sapiens.GRCh38.fa -I sample.bam -L interval1.bed -tumor sample --max-reads-per-alignment-start 0 -O sample_interval1.vcf -bamout sample_interval1.bam
gatk-4.0.5.0 --java-options "-Xmx30g" Mutect2 --native-pair-hmm-threads 20 -R ~/Homo_sapiens.GRCh38.fa -I sample.bam -L interval2.bed -tumor sample --max-reads-per-alignment-start 0 -O sample_interval2.vcf -bamout sample_interval2.bam
As a result:
-Interval1:
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT ONC1-0095
chr14 104780214 . C T . . DP=145;ECNT=1;POP_AF=5.000e-08;TLOD=269.13 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB 0/1:49,96:0.661:0,0:49,96:28:150,150:60:26:0.657,0.626,0.662:0.027,0.026,0.947
-Interval2: Nothing detected
The IGV images with the both bamouts are also provided (bamouts_igv.png).
We are using bamclipper software as a bam preprocessing step for removing primers and maybe this is interfering in the variant calling result.
We really appreciate your help if you help us to understand why we are getting these results.
-Jorge
we are working with Mutect2 to call somatic variants (snvs and indels) on targeted sequencing. We recently have noticed an issue related to an specific variant detection, so that we detect it if we have an small interval within is the variant, but if this interval is greater, it is not detected.
These are both intervals:
chr14 104780203 104780225
chr14 104780078 104780226
The interest variant is in the chr14 104780214 genomic position (within both intervals), and we are able to see it using IGV with an AF of 66%, as it is shown in the image (sample_bam_igv.png).
The command we are using is the following (we also run this with last gatk version, gatk-4.0.11.0, and have the same result):
gatk-4.0.5.0 --java-options "-Xmx30g" Mutect2 --native-pair-hmm-threads 20 -R ~/Homo_sapiens.GRCh38.fa -I sample.bam -L interval1.bed -tumor sample --max-reads-per-alignment-start 0 -O sample_interval1.vcf -bamout sample_interval1.bam
gatk-4.0.5.0 --java-options "-Xmx30g" Mutect2 --native-pair-hmm-threads 20 -R ~/Homo_sapiens.GRCh38.fa -I sample.bam -L interval2.bed -tumor sample --max-reads-per-alignment-start 0 -O sample_interval2.vcf -bamout sample_interval2.bam
As a result:
-Interval1:
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT ONC1-0095
chr14 104780214 . C T . . DP=145;ECNT=1;POP_AF=5.000e-08;TLOD=269.13 GT:AD:AF:F1R2:F2R1:MBQ:MFRL:MMQ:MPOS:SA_MAP_AF:SA_POST_PROB 0/1:49,96:0.661:0,0:49,96:28:150,150:60:26:0.657,0.626,0.662:0.027,0.026,0.947
-Interval2: Nothing detected
The IGV images with the both bamouts are also provided (bamouts_igv.png).
We are using bamclipper software as a bam preprocessing step for removing primers and maybe this is interfering in the variant calling result.
We really appreciate your help if you help us to understand why we are getting these results.
-Jorge