GATK BQSR error — Reference and BAM file chromosome name mismatch (“chr” vs. no “chr”)

Question

[iamsmor]

Hi everyone,

I'm working with the GATK pipeline (v4.5.0.0) for variant calling on human RNA-seq data aligned to GRCh38. I'm currently stuck at the BQSR (Base Quality Score Recalibration) step due to what seems to be a mismatch between my BAM file and the reference genome FASTA file.

My BAM file (Control-DMSO-24h-1.marked.bam) was generated using Homo_sapiens.GRCh38.dna.primary_assembly.fa (from Ensembl). These chromosome names are like 1, 2, MT, X, etc. (no "chr" prefix).

For BQSR, I'm using GATK's recommended Homo_sapiens_assembly38.fasta as the reference, which does have chr prefixes (chr1, chrM, etc.).

I also have known sites VCF files (dbSNP and Mills indels) provided by GATK that match the chr-prefixed reference.

When I run the GATK BQSR command, I get an error like:

gatk BaseRecalibrator \ -I /arf/scratch/semugur/markduplicates_all/Control-DMSO-24h-1.marked.bam \ -R /arf/home/semugur/Gatk/prostat/prostat_split/ref/Homo_sapiens_assembly38.fasta \ --known-sites /arf/home/semugur/Gatk/prostat/prostat_split/ref/Homo_sapiens_assembly38.dbsnp138.vcf \ --known-sites /arf/home/semugur/Gatk/prostat/prostat_split/ref/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz \ -O /arf/scratch/semugur/bqsr_prostat/Control-DMSO-24h-1_recal.table Using GATK jar /arf/home/semugur/miniconda3/envs/gatk_env/share/gatk4-4.3.0.0-0/gatk-package-4.3.0.0-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 -jar /arf/home/semugur/miniconda3/envs/gatk_env/share/gatk4-4.3.0.0-0/gatk-package-4.3.0.0-local.jar BaseRecalibrator -I /arf/scratch/semugur/markduplicates_all/Control-DMSO-24h-1.marked.bam -R /arf/home/semugur/Gatk/prostat/prostat_split/ref/Homo_sapiens_assembly38.fasta --known-sites /arf/home/semugur/Gatk/prostat/prostat_split/ref/Homo_sapiens_assembly38.dbsnp138.vcf --known-sites /arf/home/semugur/Gatk/prostat/prostat_split/ref/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz -O /arf/scratch/semugur/bqsr_prostat/Control-DMSO-24h-1_recal.table 23:36:25.769 INFO NativeLibraryLoader - Loading libgkl_compression.so from jar:file:/arf/home/semugur/miniconda3/envs/gatk_env/share/gatk4-4.3.0.0-0/gatk-package-4.3.0.0-local.jar!/com/intel/gkl/native/libgkl_compression.so 23:36:25.928 INFO BaseRecalibrator - ------------------------------------------------------------ 23:36:25.929 INFO BaseRecalibrator - The Genome Analysis Toolkit (GATK) v4.3.0.0 23:36:25.929 INFO BaseRecalibrator - For support and documentation go to https://software.broadinstitute.org/gatk/ 23:36:25.929 INFO BaseRecalibrator - Executing as semugur@arf-ui1 on Linux v5.14.0-284.30.1.el9_2.x86_64 amd64 23:36:25.929 INFO BaseRecalibrator - Java runtime: OpenJDK 64-Bit Server VM v11.0.13+7-b1751.21 23:36:25.929 INFO BaseRecalibrator - Start Date/Time: May 29, 2025 at 11:36:25 PM TRT 23:36:25.929 INFO BaseRecalibrator - ------------------------------------------------------------ 23:36:25.929 INFO BaseRecalibrator - ------------------------------------------------------------ 23:36:25.930 INFO BaseRecalibrator - HTSJDK Version: 3.0.1 23:36:25.930 INFO BaseRecalibrator - Picard Version: 2.27.5 23:36:25.930 INFO BaseRecalibrator - Built for Spark Version: 2.4.5 23:36:25.930 INFO BaseRecalibrator - HTSJDK Defaults.COMPRESSION_LEVEL : 2 23:36:25.930 INFO BaseRecalibrator - HTSJDK Defaults.USE_ASYNC_IO_READ_FOR_SAMTOOLS : false 23:36:25.930 INFO BaseRecalibrator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_SAMTOOLS : true 23:36:25.930 INFO BaseRecalibrator - HTSJDK Defaults.USE_ASYNC_IO_WRITE_FOR_TRIBBLE : false 23:36:25.930 INFO BaseRecalibrator - Deflater: IntelDeflater 23:36:25.930 INFO BaseRecalibrator - Inflater: IntelInflater 23:36:25.930 INFO BaseRecalibrator - GCS max retries/reopens: 20 23:36:25.930 INFO BaseRecalibrator - Requester pays: disabled 23:36:25.930 INFO BaseRecalibrator - Initializing engine 23:36:27.819 INFO FeatureManager - Using codec VCFCodec to read file file:///arf/home/semugur/Gatk/prostat/prostat_split/ref/Homo_sapiens_assembly38.dbsnp138.vcf 23:36:27.964 INFO FeatureManager - Using codec VCFCodec to read file file:///arf/home/semugur/Gatk/prostat/prostat_split/ref/Mills_and_1000G_gold_standard.indels.hg38.vcf.gz 23:36:28.093 INFO BaseRecalibrator - Shutting down engine [May 29, 2025 at 11:36:28 PM TRT] org.broadinstitute.hellbender.tools.walkers.bqsr.BaseRecalibrator done. Elapsed time: 0.04 minutes. Runtime.totalMemory()=2944401408 *********************************************************************** A USER ERROR has occurred: Input files reference and reads have incompatible contigs: No overlapping contigs found. reference contigs = [chr1, chr2, chr3, chr4, chr5, chr6, chr7, chr8, chr9, chr10, chr11, chr12, chr13, chr14, chr15, chr16, chr17, chr18, chr19, chr20, chr21, chr22, chrX, chrY, chrM, chr1_KI270706v1_random, chr1_KI270707v1_random, chr1_KI270708v1_random, chr1_KI270709v1_random, chr1_KI270710v1_random, chr1_KI270711v1_random,

I checked my .fai and BAM headers:

.fai from the reference has chr1, chr2, chrM, etc.

BAM header has @SQ SN:1, @SQ SN:MT, etc.

how ı can solve this problem or or should I skip to the next haplotypecaller step?

Answer 1

[Pierre Lindenbaum]

This question has been asked many times: GATK Input files reference and features have incompatible contigs: No overlapping contigs found. , GATK: Input files reads and reference have incompatible contigs: No overlapping contigs found , GATK BaseRecalibrator error: input files reference and features have incompatible contigs

you can remap your bam files to the gatk genome or you can try to rename the contigs in the different resources of the GATK bundle (fasta files, VCF files (Replacing the Chr names and position notions in vcf) )