SNP CALLING underlined high level or heterzygosity in surely highly homozygous samples
REQUIRED for all errors and issues:
a) GATK version used: 4.6.0
b) Exact command used:
c) Entire program log:
Dears,
I'm using gatk for the first times to do a propedeutial SNP calling analysis for a bulk segregant analysis in tomato. In the past, I've always used a pipeline with bcftools as caller without any problems.
I'm trying to call two samples that surely are highly homozygous in two step:
#call each sample as follow:
gatk --java-options "-Xmx4g" HaplotypeCaller --native-pair-hmm-threads 48 -R path\to\reference.fa -I file_name1.bam -O output1.g.vcf.gz -ERC GVCF
gatk --java-options "-Xmx4g" HaplotypeCaller --native-pair-hmm-threads 48 -R path\to\reference.fa -I file_name2.bam -O output2.g.vcf.gz -ERC GVCF
#recal each sample using the previous vcf in the option --alleles to visualize the homozygous for reference alleles and contrasting between two samples
gatk --java-options "-Xmx4g" HaplotypeCaller --native-pair-hmm-threads 48 -R path\to\reference.fa -I file_name1.bam --alleles output2.g.vcf.gz -O output1_recall.g.vcf.gz -ERC GVCF
gatk --java-options "-Xmx4g" HaplotypeCaller --native-pair-hmm-threads 48 -R path\to\reference.fa -I file_name2.bam --alleles output1.g.vcf.gz -O output2_recall.g.vcf.gz -ERC GVCF
#then I merged files of the same samples and converted in vcf
gatk --java-options "-Xmx96g -Xms96g" CombineGVCFs -R path\to\reference.fa --variant output1_recall.g.vcf.gz --variant output2_recall.g.vcf.gz -O combined.g.vcf.gz
gatk --java-options "-Xmx96g -Xms96g" GenotypeGVCFs -R path\to\reference.fa -V combined.g.vcf.gz -O final.vcf.gz
but the resulting vcf showed, for both samples, or heterozygous loci or homozygous ones for the alternative allele. I don't know where I wronged...
Here an example of log:
Using GATK jar /share/Tools/gatk-4.6.0.0/gatk-package-4.6.0.0-local.jar
Running:
java -Dsamjdk.use_async_io_read_samtools=false -Dsamjdk.use_async_io_write_samtools=true -Dsamjdk.use_async_io_write_tribble=false -Dsamjdk.compression_level=2 -Xmx4g -jar /share/Tools/gatk-4.6.0.0/gatk-package-4.6.0.0-local.jar HaplotypeCaller -R /home/olivierif/genome_gatk/S_lycopersicum_chromosomes.4.00.fa -I scatolone_filtered_withRG.bam -O V710213.g.vcf.gz -ERC GVCF
10:18:15.726 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/share/Tools/gatk-4.6.0.0/gatk-package-4.6.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
10:18:15.867 INFO HaplotypeCaller - ------------------------------------------------------------
10:18:15.870 INFO HaplotypeCaller - The Genome Analysis Toolkit (GATK) v4.6.0.0
10:18:15.870 INFO HaplotypeCaller - For support and documentation go to https://software.broadinstitute.org/gatk/
10:18:15.870 INFO HaplotypeCaller - Executing as olivierif@marvin on Linux v5.14.0-362.18.1.el9_3.0.1.x86_64 amd64
10:18:15.870 INFO HaplotypeCaller - Java runtime: OpenJDK 64-Bit Server VM v17.0.12+7-LTS
10:18:15.870 INFO HaplotypeCaller - Start Date/Time: October 8, 2024 at 10:18:15 AM GMT+02:00
10:18:15.870 INFO HaplotypeCaller - ------------------------------------------------------------
10:18:15.871 INFO HaplotypeCaller - ------------------------------------------------------------
10:18:15.871 INFO HaplotypeCaller - HTSJDK Version: 4.1.1
10:18:15.871 INFO HaplotypeCaller - Picard Version: 3.2.0
10:18:15.872 INFO HaplotypeCaller - Built for Spark Version: 3.5.0
10:18:15.872 INFO HaplotypeCaller - HTSJDK Defaults.COMPRESSION_LEVEL : 2
10:18:15.872 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
10:18:15.872 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
10:18:15.872 INFO HaplotypeCaller - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
10:18:15.872 INFO HaplotypeCaller - Deflater: IntelDeflater
10:18:15.872 INFO HaplotypeCaller - Inflater: IntelInflater
10:18:15.873 INFO HaplotypeCaller - GCS max retries/reopens: 20
10:18:15.873 INFO HaplotypeCaller - Requester pays: disabled
10:18:15.873 INFO HaplotypeCaller - Initializing engine
10:18:16.007 INFO HaplotypeCaller - Done initializing engine
10:18:16.009 INFO HaplotypeCallerEngine - Tool is in reference confidence mode and the annotation, the following changes will be made to any specified annotations: 'StrandBiasBySample' will be enabled. 'ChromosomeCounts', 'FisherStrand', 'StrandOddsRatio' and 'QualByDepth' annotations have been disabled
10:18:16.015 INFO NativeLibraryLoader - Loading libgkl_utils.so from jar:file:/share/Tools/gatk-4.6.0.0/gatk-package-4.6.0.0-local.jar!/com/intel/gkl/native/libgkl_utils.so
10:18:16.017 INFO NativeLibraryLoader - Loading libgkl_smithwaterman.so from jar:file:/share/Tools/gatk-4.6.0.0/gatk-package-4.6.0.0-local.jar!/com/intel/gkl/native/libgkl_smithwaterman.so
10:18:16.018 INFO IntelSmithWaterman - Using CPU-supported AVX-512 instructions
10:18:16.018 INFO SmithWatermanAligner - Using AVX accelerated SmithWaterman implementation
10:18:16.021 INFO HaplotypeCallerEngine - Standard Emitting and Calling confidence set to -0.0 for reference-model confidence output
10:18:16.021 INFO HaplotypeCallerEngine - All sites annotated with PLs forced to true for reference-model confidence output
10:18:16.029 INFO NativeLibraryLoader - Loading libgkl_pairhmm_omp.so from jar:file:/share/Tools/gatk-4.6.0.0/gatk-package-4.6.0.0-local.jar!/com/intel/gkl/native/libgkl_pairhmm_omp.so
10:18:16.044 INFO IntelPairHmm - Using CPU-supported AVX-512 instructions
10:18:16.044 INFO IntelPairHmm - Flush-to-zero (FTZ) is enabled when running PairHMM
10:18:16.045 INFO IntelPairHmm - Available threads: 96
10:18:16.045 INFO IntelPairHmm - Requested threads: 4
10:18:16.045 INFO PairHMM - Using the OpenMP multi-threaded AVX-accelerated native PairHMM implementation
10:18:16.086 INFO ProgressMeter - Starting traversal
10:18:16.086 INFO ProgressMeter - Current Locus Elapsed Minutes Regions Processed Regions/Minute
10:18:16.423 WARN InbreedingCoeff - InbreedingCoeff will not be calculated at position SL4.0ch00:7054 and possibly subsequent; at least 10 samples must have called genotypes
10:18:21.567 WARN DepthPerSampleHC - Annotation will not be calculated at position SL4.0ch00:278145 and possibly subsequent; genotype for sample V710213 is not called
10:18:21.567 WARN StrandBiasBySample - Annotation will not be calculated at position SL4.0ch00:278145 and possibly subsequent; genotype for sample V710213 is not called
10:18:26.301 INFO ProgressMeter - SL4.0ch00:389772 0.2 1580 9281.4
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02:23:04.266 INFO HaplotypeCaller - 1356164 read(s) filtered by: MappingQualityReadFilter
0 read(s) filtered by: MappingQualityAvailableReadFilter
0 read(s) filtered by: MappedReadFilter
0 read(s) filtered by: NotSecondaryAlignmentReadFilter
0 read(s) filtered by: NotDuplicateReadFilter
0 read(s) filtered by: PassesVendorQualityCheckReadFilter
0 read(s) filtered by: NonZeroReferenceLengthAlignmentReadFilter
0 read(s) filtered by: GoodCigarReadFilter
0 read(s) filtered by: WellformedReadFilter
1356164 total reads filtered out of 142445698 reads processed
02:23:04.266 INFO ProgressMeter - SL4.0ch12:66686701 569.1 2608404 4583.8
02:23:04.266 INFO ProgressMeter - Traversal complete. Processed 2608404 total regions in 569.1 minutes.
02:23:04.321 INFO VectorLoglessPairHMM - Time spent in setup for JNI call : 25.408589052
02:23:04.322 INFO PairHMM - Total compute time in PairHMM computeLogLikelihoods() : 914.1325713330001
02:23:04.322 INFO SmithWatermanAligner - Total compute time in native Smith-Waterman : 825.93 sec
02:24:59.760 INFO HaplotypeCaller - Shutting down engine
[October 11, 2024 at 2:24:59 AM GMT+02:00] org.broadinstitute.hellbender.tools.walkers.haplotypecaller.HaplotypeCaller done. Elapsed time: 570.99 minutes.
Runtime.totalMemory()=566231040
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I believe the second call step is totally unnecessary since that removes all the remaining reference confidence sites for your cause.
Instead of recalling (which we also think is problematic. We do not recommend using GVCF files as allele inputs for HaplotypeCaller since that file is still raw and not genotyped), we recommend combining the first 2 GVCF files and joint genotyping that file. The resulting genotype file will contain HOMREF, HOMVAR and HET loci for all common loci that those 2 samples can convene.
I hope this helps.
Regards.
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I am here to seek your suggestion rather than solving your issue. I am also trying to run GATK for 'tomato'. I am performing Bulked Segregant Analysis in tomato. I am stuck at the 'BQSR (Base Quality Score Recalibration)' step because I cannot not find 'known sites' for tomato. Could you please suggest where I can find this information or datasets to access and/or download?
Thank you,
Navin Shrestha
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