Dear GATK,

I have used the gatk BaseRecalibrator tool on my bam files but I noticed that all the  Recal_data.tables   that have been generated on my 97 bam files have exactly the same weight

 From 111005 bytes and 111100, this seems very strange , I have processed other samples and the Recal_data.table files were always very variable in size.

For recalibration I’ve used the following files from the GATK bundle :

--known-sites Homo_sapiens_assembly38.known_indels.vcf --known-sites

--known-sites 1000G_phase1.snps.high_confidence.hg38.vcf --known-sites --known-sites

--known-sites  Mills_and_1000G_gold_standard.indels.hg38.vcf

The only thing that I changed in  my pipeline compared to the past  was the fasta file that I used to align the fastq files  to the reference genome. Before I used the fasta hg38 available in  GATK bundle, this time I used the fasta file that  did not contain the ALT haplotypes https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.26_GRCh38/GRCh38_major_release_seqs_for_alignment_pipelines/

Are the known sites compatible with my new fasta file (hg38)?

REQUIRED for all errors and issues:
a) GATK version used:The Genome Analysis Toolkit (GATK) v4.2.3.0

b) Exact command used:

/lustrehome/sharonnatashacox/.conda/envs/venv2/bin/gatk BaseRecalibrator -R /lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/NO_ALT_GENOME/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna -I  /lustre/parkinsongiudice/PESCE/110_EGF/OUT_sorted_nodup_prefix.bam -O Recal_data110_manua,table --known-sites /lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/GATKbundle/Homo_sapiens_assembly38.known_indels.vcf --known-sites /lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/GATKbundle/1000G_phase1.snps.high_confidence.hg38.vcf --known-sites /lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/GATKbundle/Mills_and_1000G_gold_standard.indels.hg38.vcf --java-options -DGATK_STACKTRACE_ON_USER_EXCEPTION=true; done
 
c) Entire program log:

/OUT_sorted_nodup_prefix.bam
Using GATK jar /lustrehome/sharonnatashacox/.conda/envs/venv2/share/gatk4-4.2.3.0-1/gatk-package-4.2.3.0-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 -DGATK_STACKTRACE_ON_USER_EXCEPTION=true -jar /lustrehome/sharonnatashacox/.conda/envs/venv2/share/gatk4-4.2.3.0-1/gatk-package-4.2.3.0-local.jar BaseRecalibrator -R /lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/NO_ALT_GENOME/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna -I /lustre/parkinsongiudice/PESCE/110_EGF/OUT_sorted_nodup_prefix.bam -O /lustre/parkinsongiudice/PESCE/110_EGF/Recal_data.table --known-sites /lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/GATKbundle/Homo_sapiens_assembly38.known_indels.vcf --known-sites /lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/GATKbundle/1000G_phase1.snps.high_confidence.hg38.vcf --known-sites /lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/GATKbundle/Mills_and_1000G_gold_standard.indels.hg38.vcf
17:32:55.690 INFO  NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/lustrehome/sharonnatashacox/.conda/envs/venv2/share/gatk4-4.2.3.0-1/gatk-package-4.2.3.0-local.jar!/com/intel/gkl/native/libgkl_compression.so
Jul 22, 2022 5:32:55 PM shaded.cloud_nio.com.google.auth.oauth2.ComputeEngineCredentials runningOnComputeEngine
INFO: Failed to detect whether we are running on Google Compute Engine.
17:32:55.958 INFO  BaseRecalibrator - ------------------------------------------------------------
17:32:55.959 INFO  BaseRecalibrator - The Genome Analysis Toolkit (GATK) v4.2.3.0
17:32:55.959 INFO  BaseRecalibrator - For support and documentation go to https://software.broadinstitute.org/gatk/
17:32:55.959 INFO  BaseRecalibrator - Executing as sharonnatashacox@ui02.recas.ba.infn.it on Linux v3.10.0-1160.71.1.el7.x86_64 amd64
17:32:55.959 INFO  BaseRecalibrator - Java runtime: OpenJDK 64-Bit Server VM v11.0.8-internal+0-adhoc..src
17:32:55.959 INFO  BaseRecalibrator - Start Date/Time: July 22, 2022 at 5:32:55 PM CEST
17:32:55.960 INFO  BaseRecalibrator - ------------------------------------------------------------
17:32:55.960 INFO  BaseRecalibrator - ------------------------------------------------------------
17:32:55.961 INFO  BaseRecalibrator - HTSJDK Version: 2.24.1
17:32:55.961 INFO  BaseRecalibrator - Picard Version: 2.25.4
17:32:55.961 INFO  BaseRecalibrator - Built for Spark Version: 2.4.5
17:32:55.961 INFO  BaseRecalibrator - HTSJDK Defaults.COMPRESSION_LEVEL : 2
17:32:55.961 INFO  BaseRecalibrator - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false
17:32:55.961 INFO  BaseRecalibrator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true
17:32:55.961 INFO  BaseRecalibrator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false
17:32:55.962 INFO  BaseRecalibrator - Deflater: IntelDeflater
17:32:55.962 INFO  BaseRecalibrator - Inflater: IntelInflater
17:32:55.962 INFO  BaseRecalibrator - GCS max retries/reopens: 20
17:32:55.962 INFO  BaseRecalibrator - Requester pays: disabled
17:32:55.962 INFO  BaseRecalibrator - Initializing engine
17:32:56.615 INFO  FeatureManager - Using codec VCFCodec to read file file:///lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/GATKbundle/Homo_sapiens_assembly38.known_indels.vcf
17:32:56.755 INFO  FeatureManager - Using codec VCFCodec to read file file:///lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/GATKbundle/1000G_phase1.snps.high_confidence.hg38.vcf
17:32:57.266 INFO  FeatureManager - Using codec VCFCodec to read file file:///lustrehome/sharonnatashacox/ex_storage_2021/Chromosomes/GATKbundle/Mills_and_1000G_gold_standard.indels.hg38.vcf
17:32:57.456 INFO  BaseRecalibrator - Done initializing engine
17:32:57.461 INFO  BaseRecalibrationEngine - The covariates being used here: 
17:32:57.461 INFO  BaseRecalibrationEngine -     ReadGroupCovariate
17:32:57.461 INFO  BaseRecalibrationEngine -     QualityScoreCovariate
17:32:57.461 INFO  BaseRecalibrationEngine -     ContextCovariate
17:32:57.461 INFO  BaseRecalibrationEngine -     CycleCovariate
17:32:57.470 INFO  ProgressMeter - Starting traversal
17:32:57.471 INFO  ProgressMeter -        Current Locus  Elapsed Minutes       Reads Processed     Reads/Minute
17:33:07.509 INFO  ProgressMeter -         chr1:6072168              0.2                177000        1058296.0
17:33:17.532 INFO  ProgressMeter -        chr1:11112939              0.3                382000        1142572.3......................................................................................................................................................

18:32:17.262 INFO  ProgressMeter -       chrX:153949283             59.3              72859000        1228032.8
18:32:27.295 INFO  ProgressMeter - chr22_KI270734v1_random:82127             59.5              73109000        1228783.3
18:32:29.924 WARN  IntelInflater - Zero Bytes Written : 0
18:32:29.927 INFO  BaseRecalibrator - 5103426 read(s) filtered by: MappingQualityNotZeroReadFilter 
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: WellformedReadFilter 
5103426 total reads filtered
18:32:29.927 INFO  ProgressMeter - chrUn_GL000218v1:160879             59.5              73176003        1229003.3
18:32:29.927 INFO  ProgressMeter - Traversal complete. Processed 73176003 total reads in 59.5 minutes.
18:32:29.999 INFO  BaseRecalibrator - Calculating quantized quality scores...
18:32:30.012 INFO  BaseRecalibrator - Writing recalibration report...
18:32:30.814 INFO  BaseRecalibrator - ...done!
18:32:30.814 INFO  BaseRecalibrator - BaseRecalibrator was able to recalibrate 73176003 reads
18:32:30.814 INFO  BaseRecalibrator - Shutting down engine
[July 22, 2022 at 6:32:30 PM CEST] org.broadinstitute.hellbender.tools.walkers.bqsr.BaseRecalibrator done. Elapsed time: 59.59 minutes.
Runtime.totalMemory()=1581252608
Tool returned:
SUCCESS

Thankyou for your help