Hi,

I've tried to annotate denovo variants in my TRIO samples (Mother, Father, Proband) vcf using VariantAnnotator PossibleDenovo. However, none of my variants were annotated with either hiConfDeNovo or loConfDeNovo. 

I manually checked some of the variants, and they should pass the crieteria for either hiConfDeNovo or loConfDeNovo variants.

What could went wrong here, and how can I fix it? Thanks in advance !!!

Below are the command I used to merge the gvcf and run VariantAnnotator PossibleDenovo

1) Merge gvcfs (called by GATK haplotypecaller-done by my colleague) 

(gatk) root@2dc5158253fb:/gatk/my_data# gatk CombineGVCFs -R Homo_sapiens_assembly38.fasta --variant GGSpi006.g.vcf.gz --variant GGSpi007.g.vcf.gz --variant GGSpi005.g.vcf.gz -O GGSpi005.cohort.GATKpipeline.g.vcf.gz

2) The genotypes were missing after I merged the gvcfs, so after reading some guide on GATK forum, I perform joint genotyping

(gatk) root@2dc5158253fb:/gatk/my_data# gatk GenotypeGVCFs -R Homo_sapiens_assembly38.fasta -V GGSpi005.cohort.GATKpipeline.g.vcf.gz -O GGSpi005.cohort.GATKpipeline.combineGT.g.vcf.gz

3) Ran VariantAnnotator

(gatk) root@2dc5158253fb:/gatk/my_data# gatk VariantAnnotator -A PossibleDeNovo  -R Homo_sapiens_assembly38.fasta --pedigree GGSpi005.ped -V GGSpi005.cohort.GATKpipeline.combineGT.g.vcf.gz -O GGSpi005.cohort.GATKpipeline.combineGT.possibledenovo.g.vcf.gz

Logs from VariantAnnotator:

08:09:36.135 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/gatk/gatk-package-4.1.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.so

Sep 02, 2021 8:09:37 AM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine

INFO: Failed to detect whether we are running on Google Compute Engine.

08:09:37.841 INFO  VariantAnnotator - ------------------------------------------------------------

08:09:37.841 INFO  VariantAnnotator - The Genome Analysis Toolkit (GATK) v4.1.3.0

08:09:37.842 INFO  VariantAnnotator - For support and documentation go to https://software.broadinstitute.org/gatk/

08:09:37.842 INFO  VariantAnnotator - Executing as root@2dc5158253fb on Linux v4.15.0-128-generic amd64

08:09:37.842 INFO  VariantAnnotator - Java runtime: OpenJDK 64-Bit Server VM v1.8.0_191-8u191-b12-0ubuntu0.16.04.1-b12

08:09:37.842 INFO  VariantAnnotator - Start Date/Time: September 2, 2021 8:09:36 AM UTC

08:09:37.842 INFO  VariantAnnotator - ------------------------------------------------------------

08:09:37.842 INFO  VariantAnnotator - ------------------------------------------------------------

08:09:37.843 INFO  VariantAnnotator - HTSJDK Version: 2.20.1

08:09:37.843 INFO  VariantAnnotator - Picard Version: 2.20.5

08:09:37.843 INFO  VariantAnnotator - HTSJDK Defaults.COMPRESSION_LEVEL : 2

08:09:37.843 INFO  VariantAnnotator - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false

08:09:37.843 INFO  VariantAnnotator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true

08:09:37.843 INFO  VariantAnnotator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false

08:09:37.843 INFO  VariantAnnotator - Deflater: IntelDeflater

08:09:37.843 INFO  VariantAnnotator - Inflater: IntelInflater

08:09:37.843 INFO  VariantAnnotator - GCS max retries/reopens: 20

08:09:37.843 INFO  VariantAnnotator - Requester pays: disabled

08:09:37.844 WARN  VariantAnnotator -

 

   !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

 

   Warning: VariantAnnotator is a BETA tool and is not yet ready for use in production

 

   !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

 

 

08:09:37.844 INFO  VariantAnnotator - Initializing engine

08:09:38.286 INFO  FeatureManager - Using codec VCFCodec to read file file:///gatk/my_data/GGSpi005.cohort.GATKpipeline.combineGT.g.vcf.gz

08:09:38.647 INFO  VariantAnnotator - Done initializing engine

08:09:38.735 INFO  ProgressMeter - Starting traversal

08:09:38.735 INFO  ProgressMeter -        Current Locus  Elapsed Minutes    Variants Processed  Variants/Minute

08:09:39.031 INFO  PedReader - Reading PED file GGSpi005.ped with missing fields: []

08:09:39.033 INFO  PedReader - Phenotype is other? false

08:09:48.749 INFO  ProgressMeter -        chr1:80703442              0.2                185000        1108558.9

08:09:58.782 INFO  ProgressMeter -       chr1:182224598              0.3                396000        1185273.9

08:10:08.787 INFO  ProgressMeter -        chr2:16645232              0.5                599000        1195927.1

08:10:18.835 INFO  ProgressMeter -       chr2:107532026              0.7                815000        1219451.4

08:10:28.869 INFO  ProgressMeter -       chr2:213961585              0.8               1043000        1248254.7

08:10:38.896 INFO  ProgressMeter -        chr3:70258454              1.0               1279000        1275577.2

08:10:48.922 INFO  ProgressMeter -       chr3:157701073              1.2               1485000        1269465.9

08:10:58.955 INFO  ProgressMeter -        chr4:43128759              1.3               1695000        1267779.5

08:11:08.979 INFO  ProgressMeter -       chr4:138932992              1.5               1926000        1280528.3

08:11:18.997 INFO  ProgressMeter -        chr5:36332061              1.7               2158000        1291416.5

08:11:29.007 INFO  ProgressMeter -       chr5:152626381              1.8               2402000        1306950.1

08:11:39.033 INFO  ProgressMeter -        chr6:51516372              2.0               2634000        1313737.6

08:11:49.046 INFO  ProgressMeter -       chr6:151071223              2.2               2853000        1313636.7

08:11:59.047 INFO  ProgressMeter -        chr7:56735293              2.3               3060000        1308512.5

08:12:09.077 INFO  ProgressMeter -          chr8:343368              2.5               3307000        1319790.9

08:12:19.094 INFO  ProgressMeter -       chr8:103320886              2.7               3556000        1330514.7

08:12:29.115 INFO  ProgressMeter -        chr9:42375718              2.8               3774000        1329029.2

08:12:39.134 INFO  ProgressMeter -       chr10:17793490              3.0               4016000        1335705.9

08:12:49.136 INFO  ProgressMeter -      chr10:112786823              3.2               4256000        1341169.4

08:12:59.172 INFO  ProgressMeter -       chr11:71343184              3.3               4499000        1346757.3

08:13:09.192 INFO  ProgressMeter -       chr12:32329105              3.5               4740000        1351351.4

08:13:19.200 INFO  ProgressMeter -       chr13:16124482              3.7               4979000        1355045.0

08:13:29.228 INFO  ProgressMeter -      chr13:109140124              3.8               5225000        1360128.1

08:13:39.261 INFO  ProgressMeter -       chr15:23086088              4.0               5468000        1364016.2

08:13:49.293 INFO  ProgressMeter -       chr16:17065339              4.2               5715000        1368550.9

08:13:59.313 INFO  ProgressMeter -       chr17:32971285              4.3               5959000        1372103.6

08:14:09.322 INFO  ProgressMeter -       chr18:56553291              4.5               6202000        1375237.4

08:14:19.355 INFO  ProgressMeter -        chr20:4817194              4.7               6447000        1378447.7

08:14:29.358 INFO  ProgressMeter -       chr21:31112171              4.8               6692000        1381583.7

08:14:39.391 INFO  ProgressMeter -        chrX:56103925              5.0               6939000        1384772.0

08:14:45.800 INFO  ProgressMeter -        chrY:56863546              5.1               7089754        1385330.9

08:14:45.800 INFO  ProgressMeter - Traversal complete. Processed 7089754 total variants in 5.1 minutes.

08:14:45.966 INFO  VariantAnnotator - Shutting down engine

[September 2, 2021 8:14:45 AM UTC] org.broadinstitute.hellbender.tools.walkers.annotator.VariantAnnotator done. Elapsed time: 5.16 minutes.

Runtime.totalMemory()=1352138752

GATK was run in a docker container: 

docker run -v ~/my_project:/gatk/my_data -it broadinstitute/gatk:4.1.3.0

Pedigree file:

FAM1 GGSpi006 0 0 1 1

FAM2 GGSpi007 0 0 2 1

FAM1 GGSpi005 GGSpi006 GGspi007 1 2

Some relevant header in the output header:

##GATKCommandLine=<ID=CombineGVCFs,CommandLine="CombineGVCFs  --output GGSpi005.cohort.GATKpipeline.g.vcf.gz --variant GGSpi006.g.vcf.gz --variant GGSpi007.g.vcf.gz --variant GGSpi005.g.vcf.gz --reference Homo_sapiens_assembly38.fasta  --convert-to-base-pair-resolution false --break-bands-at-multiples-of 0 --input

##GATKCommandLine=<ID=GenotypeGVCFs,CommandLine="GenotypeGVCFs  --output GGSpi005.cohort.GATKpipeline.combineGT.g.vcf.gz --variant GGSpi005.cohort.GATKpipeline.g.vcf.gz --reference Homo_sapiens_assembly38.fasta  --include-non-variant-sites false --merge-input-intervals false --input-is-somatic false --tumor-lod-to

##GATKCommandLine=<ID=HaplotypeCaller,CommandLine="HaplotypeCaller  --emit-ref-confidence GVCF --gvcf-gq-bands 10 --gvcf-gq-bands 20 --gvcf-gq-bands 30 --gvcf-gq-bands 40 --gvcf-gq-bands 50 --gvcf-gq-bands 60 --gvcf-gq-bands 70 --gvcf-gq-bands 80 --gvcf-gq-bands 90 --use-new-qual-calculator true --contamination-fr

##INFO=<ID=AC,Number=A,Type=Integer,Description="Allele count in genotypes, for each ALT allele, in the same order as listed">

##INFO=<ID=AF,Number=A,Type=Float,Description="Allele Frequency, for each ALT allele, in the same order as listed">

##INFO=<ID=AN,Number=1,Type=Integer,Description="Total number of alleles in called genotypes">

##INFO=<ID=AS_InbreedingCoeff,Number=A,Type=Float,Description="allele specific heterozygosity as estimated from the genotype likelihoods per-sample when compared against the Hardy-Weinberg expectation; relate to inbreeding coefficient">

##INFO=<ID=AS_QD,Number=A,Type=Float,Description="Allele-specific Variant Confidence/Quality by Depth">

##INFO=<ID=AS_RAW_BaseQRankSum,Number=1,Type=String,Description="raw data for allele specific rank sum test of base qualities">

##INFO=<ID=AS_RAW_MQ,Number=1,Type=String,Description="Allele-specfic raw data for RMS Mapping Quality">

##INFO=<ID=AS_RAW_MQRankSum,Number=1,Type=String,Description="Allele-specfic raw data for Mapping Quality Rank Sum">

##INFO=<ID=AS_RAW_ReadPosRankSum,Number=1,Type=String,Description="allele specific raw data for rank sum test of read position bias">

##INFO=<ID=AS_SB_TABLE,Number=1,Type=String,Description="Allele-specific forward/reverse read counts for strand bias tests">

##INFO=<ID=BaseQRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt Vs. Ref base qualities">

##INFO=<ID=DP,Number=1,Type=Integer,Description="Approximate read depth; some reads may have been filtered">

##INFO=<ID=DS,Number=0,Type=Flag,Description="Were any of the samples downsampled?">

##INFO=<ID=END,Number=1,Type=Integer,Description="Stop position of the interval">

##INFO=<ID=ExcessHet,Number=1,Type=Float,Description="Phred-scaled p-value for exact test of excess heterozygosity">

##INFO=<ID=FS,Number=1,Type=Float,Description="Phred-scaled p-value using Fisher's exact test to detect strand bias">

##INFO=<ID=InbreedingCoeff,Number=1,Type=Float,Description="Inbreeding coefficient as estimated from the genotype likelihoods per-sample when compared against the Hardy-Weinberg expectation">

##INFO=<ID=MLEAC,Number=A,Type=Integer,Description="Maximum likelihood expectation (MLE) for the allele counts (not necessarily the same as the AC), for each ALT allele, in the same order as listed">

##INFO=<ID=MLEAF,Number=A,Type=Float,Description="Maximum likelihood expectation (MLE) for the allele frequency (not necessarily the same as the AF), for each ALT allele, in the same order as listed">

##INFO=<ID=MQ,Number=1,Type=Float,Description="RMS Mapping Quality">

##INFO=<ID=MQRankSum,Number=1,Type=Float,Description="Z-score From Wilcoxon rank sum test of Alt vs. Ref read mapping qualities">

##INFO=<ID=QD,Number=1,Type=Float,Description="Variant Confidence/Quality by Depth">

##INFO=<ID=RAW_MQandDP,Number=2,Type=Integer,Description="Raw data (sum of squared MQ and total depth) for improved RMS Mapping Quality calculation. Incompatible with deprecated RAW_MQ formulation.">

##INFO=<ID=ReadPosRankSum,Number=1,Type=Float,Description="Z-score from Wilcoxon rank sum test of Alt vs. Ref read position bias">

##INFO=<ID=SOR,Number=1,Type=Float,Description="Symmetric Odds Ratio of 2x2 contingency table to detect strand bias">

##INFO=<ID=hiConfDeNovo,Number=1,Type=String,Description="High confidence possible de novo mutation (GQ >= 20 for all trio members)=[comma-delimited list of child samples]">

##INFO=<ID=loConfDeNovo,Number=1,Type=String,Description="Low confidence possible de novo mutation (GQ >= 10 for child, GQ > 0 for parents)=[comma-delimited list of child samples]">

##reference=file:///gatk/my_data/Homo_sapiens_assembly38.fasta

##source=CombineGVCFs

##source=GenotypeGVCFs

##source=HaplotypeCaller

Some output:

#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT GGSpi005 GGSpi006 GGSpi007

chr1    72078   .       A       C       76.50   .       AC=2;AF=0.333;AN=6;DP=16;ExcessHet=0.4576;FS=0.000;MLEAC=1;MLEAF=0.167;MQ=29.00;QD=29.56;SOR=2.303      GT:AD:DP:GQ:PGT:PID:PL  0/0:7,0:7:21:.:.:0,21,203       0/0:7,0:7:21:.:.:0,21,223       1/1:0,2:2:6:1|1:72078_A_C:90,6,0

chr1    85222   .       A       T       35.48   .       AC=2;AF=0.333;AN=6;DP=23;ExcessHet=0.4576;FS=0.000;MLEAC=1;MLEAF=0.167;MQ=21.00;QD=17.74;SOR=2.303      GT:AD:DP:GQ:PL  0/0:8,0:8:21:0,21,248   0/0:13,0:13:36:0,36,452 1/1:0,2:2:6:49,6,0

chr1    183598  .       C       G       57.58   .       AC=1;AF=0.250;AN=4;BaseQRankSum=1.58;DP=122;ExcessHet=3.0103;FS=3.444;MLEAC=1;MLEAF=0.250;MQ=40.04;MQRankSum=-2.817e+00;QD=0.89;ReadPosRankSum=1.22;SOR=1.477   GT:AD:DP:GQ:PL  0/1:57,8:65:65:65,0,1604        0/0:57,0:57:32:0,32,1533        ./.:0,0:0:.:0,0,0