Good afternoon,

I'm using GATK 4.1.7.0 and I'm trying to apply the Haplotypecaller function to a bam file. I was following mainly this post (https://gatk.broadinstitute.org/hc/en-us/articles/360039568932--How-to-Map-and-clean-up-short-read-sequence-data-efficiently) and from that bam file I'm trying to create the GVCF file. Here is my command:

java -jar ~/softwares/GATKK/gatk/gatk-package-4.1.7.0-local.jar HaplotypeCaller --reference Pmuralis_1.0.fa --input mergeandaligned.bam --output mergeandaligned.g.vcf.gz -A StrandBiasBySample -ERC GVCF

I was previously doing the command without the StrandBiasBySample but i saw here (https://gatkforums.broadinstitute.org/gatk/discussion/6813/several-annotations-not-working-in-gatk-haplotype-caller) that was recommended, but I still have the same problem

 

When I obtain the output, in the log file I have these warnings:

    14:54:38.207 WARN StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null
    14:54:38.658 WARN StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null
    14:54:38.659 WARN StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null
    14:54:39.609 WARN StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null
    14:54:39.610 WARN StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null
    14:54:41.794 WARN StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null
    14:54:42.038 WARN StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null
    14:54:42.038 WARN StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null
    14:54:43.355 INFO ProgressMeter - Podmur_chrom0001:897923 0.2 3250 19476.6

 

Later, when I explore the obtained file, I see that for most of the observations, the ALT file is missing, and is as <NON_REF>, as you can see here:

    #CHROM	POS	ID	REF	ALT	QUAL	FILTER	INFO	FORMAT	WA01_1
    Podmur_chrom0001	1	.	T	<NON_REF>	.	.	END=17	GT:DP:GQ:MIN_DP:PL	0/0:0:0:0:0,0,0
    Podmur_chrom0001	18	.	T	<NON_REF>	.	.	END=47	GT:DP:GQ:MIN_DP:PL	0/0:1:3:1:0,3,37
    Podmur_chrom0001	48	.	G	<NON_REF>	.	.	END=48	GT:DP:GQ:MIN_DP:PL	0/0:1:0:1:0,0,0
    P

I did not do the next step because I want to solve this first. Thank you very much!

> Later, when I explore the obtained file, I see that for most of the observations, the ALT file is missing, and is as <non_ref>, as you can see here:


because you are calling in **GVCF** mode  (` -ERC GVCF`) : https://gatkforums.broadinstitute.org/gatk/discussion/4017/what-is-a-gvcf-and-how-is-it-different-from-a-regular-vcf