StrandBiasBySample error Haplotypecaller

Question

[gubrins]

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!

[liwujun23]

hello, i met the same problem with , so how do you perfrom the next step? thanks !looking forward your reply!

Answer 1

[Pierre Lindenbaum]

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

[gubrins]

Thank you very much for your help Pierre, I understand from there that my output was expected then. I was worried for all the WARNINGS I was getting. If it's not a problem, can you confirm me what do I have to do after getting the GVCF file? I have to combine my different GVCF files and then use GenotypeGVCF, right? My goal is to phase the combined vcf file with beagle, if I don't have problems. Do you know if the phasing is possible with GATK?

[Pierre Lindenbaum]

These are just warning. I think it's because you have some sites with a very low depth.

[liwujun23]

So do you think that is ignored? Thank you !

[gubrins]

Heys, yes, as Pierre said those errors are normal!

[1549227291]

hello , Pierre ! I meet the same question using GATK4.1.9.0 , I am a fresh . Could you help me ? Thanks very much! Here is my command: gatk HaplotypeCaller -R /home/variation/input/NCTC11134/fasta/N.far_NCTC11134.fa --emit-ref-confidence GVCF -I CD142.sorted.markdup.bam -O CD142.g.vcf

i don' know why it reported the error , such as DepthPerSampleHC and StrandBiasBySample , Could you explain them ? I need your help specially.

here are errors:

16:06:46.622 WARN  DepthPerSampleHC - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null


16:06:46.623 WARN  StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null

16:06:46.623 WARN  StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null

16:06:46.623 WARN  StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null

16:06:46.624 WARN  StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null

16:06:46.624 WARN  StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null

16:06:46.624 WARN  StrandBiasBySample - Annotation will not be calculated, genotype is not called or alleleLikelihoodMap is null


16:06:50.918 INFO  ProgressMeter - NZ_LN868938.1:634261              3.5                  4090           1182.7

16:07:01.275 INFO  ProgressMeter - NZ_LN868938.1:659185              3.6                  4290           1181.6