Variant calling and QC

Note: if the following error appears:
Error occurred during initialization of VM
Could not reserve enough space for 50331648KB object heap

reduce memory by adding --mem=10 OR --mem=8 to the analysis_commands.txt or initial command
change time: --time 72:00:00 to initial command
start from step already finished when cut-off early (make sure to use the same script as previously) add: "-s build" to analysis_commands txt file

Check reference file to be used for variant calling. Are the gVCF contigs named chr1 or 1? choose the appropriate reference:
resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta -> for chr1, chr2 etc
Homo_sapiens.GRCh38.dna_sm.primary_assembly -> for 1,2,3 etc (CD genomics gVCFs)

examples

Run annotation only

BGI BATCH9

NAME.vcf.gz

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb submit_csg
--cmd_file analysis_commands_BGI_Batch9_ANN.txt

analysis_commands_BGI_Batch9_ANN.txt

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb annovar
--container-option /mnt/mfs/statgen/containers/gatk4-annovar.sif
--vcf-prefix /mnt/mfs/statgen/isabelle/BGI_BATCH9_BGISEQ_extra/combine/result_variation/combine
--keep "splic|exonic" --humandb /mnt/mfs/statgen/isabelle/REF/humandb
--x-ref /mnt/mfs/statgen/isabelle/REF/humandb/mart_export_2021_LOFtools.txt

Run joint calling

SA2 batch

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb submit_csg
--cmd_file analysis_commands_SA2.txt

analysis_commands_SA2.txt

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb call
--container-option /mnt/mfs/statgen/containers/gatk4-annovar.sif
--vcf-prefix /mnt/mfs/statgen/isabelle/African_BAM/African_VCF_files/SA2/Merged/SA2
--samples /mnt/mfs/statgen/isabelle/African_BAM/African_VCF_files/SA2/SA2.txt
--samples-dir /mnt/mfs/statgen/isabelle/African_BAM/African_VCF_files/SA2/SA2/
--ref-genome /mnt/mfs/statgen/isabelle/REF/refs/Homo_sapiens.GRCh37.75.dna_sm.primary_assembly.fa

Full EXAMPLE

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb call
--container-option /mnt/mfs/statgen/containers/gatk4-annovar.sif
--vcf-prefix output/minimal_example
--samples /mnt/mfs/statgen/data_private/gatk_joint_call_example/20200820_sample_manifest.txt
--samples-dir /mnt/mfs/statgen/data_private/gatk_joint_call_example/
--ref-genome /mnt/mfs/statgen/isabelle/REF/refs/Homo_sapiens.GRCh37.75.dna_sm.primary_assembly.fa

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb filter
--container-option /mnt/mfs/statgen/containers/gatk4-annovar.sif
--vcf-prefix output/minimal_example

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb annovar
--container-option /mnt/mfs/statgen/containers/gatk4-annovar.sif
--vcf-prefix output/minimal_example.snp_indel.filter.PASS
--keep "splic|exonic"
--humandb /mnt/mfs/statgen/isabelle/REF/humandb
--x-ref /mnt/mfs/statgen/isabelle/REF/humandb/mart_export_2019_LOFtools3.txt

To skip step 2 (filter) -> Output name of RAW file is vcf-prefix name

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb call
--container-option /mnt/mfs/statgen/containers/gatk4-annovar.sif
--vcf-prefix output/minimal_example
--samples /mnt/mfs/statgen/data_private/gatk_joint_call_example/20200820_sample_manifest.txt
--samples-dir /mnt/mfs/statgen/data_private/gatk_joint_call_example/
--ref-genome /mnt/mfs/statgen/isabelle/REF/refs/Homo_sapiens.GRCh37.75.dna_sm.primary_assembly.fa

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb annovar
--container-option /mnt/mfs/statgen/containers/gatk4-annovar.sif
--vcf-prefix output/minimal_example --keep "splic|exonic"
--humandb /mnt/mfs/statgen/isabelle/REF/humandb
--x-ref /mnt/mfs/statgen/isabelle/REF/humandb/mart_export_2019_LOFtools3.txt

Update memory

sos run ~/gatk_joint_calling/gatk_joint_calling.ipynb submit_csg --cmd_file analysis_commands_CDgenomic_batch1_051622.txt --mem 64

Example analysis_commands.txt with new pipeline and basic filter

module load Singularity module load VCFTOOLS/0.1.17 module load PLINK/1.9.10 module load TABIX/1.6 sos run ~/bioworkflows/variant-calling/gatk_joint_calling.ipynb call
--container-option /mnt/vast/hpc/csg/containers/gatk4-annovar.sif
--vcf-prefix /mnt/vast/hpc/csg/Thashi/Wave4_AFRbatch3_2/AFRBatch3_2_Joint_calling/Wave4_AfrBatch3_2
--samples /mnt/vast/hpc/csg/Thashi/Wave4_AFRbatch3_2/AFRBatch3_2/Samples_Wave4_AFRbatch3_2.txt
--samples-dir /mnt/vast/hpc/csg/Thashi/Wave4_AFRbatch3_2/AFRBatch3_2/
--ref-genome /mnt/vast/hpc/csg/isabelle/REF/refs/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta
--cwd /mnt/vast/hpc/csg/Thashi/Wave4_AFRbatch3_2/AFRBatch3_2_Joint_calling/
--vcf-filter basic

sos run ~/bioworkflows/variant-calling/gatk_joint_calling.ipynb basic_filter
--container-option /mnt/vast/hpc/csg/containers/gatk4-annovar.sif
--vcf-prefix /mnt/vast/hpc/csg/Thashi/Wave4_AFRbatch3_2/AFRBatch3_2_Joint_calling/Wave4_AfrBatch3_2
--ref-genome /mnt/vast/hpc/csg/isabelle/REF/refs/resources_broad_hg38_v0_Homo_sapiens_assembly38.fasta
--cwd /mnt/vast/hpc/csg/Thashi/Wave4_AFRbatch3_2/AFRBatch3_2_Joint_calling/
--vcf-filter basic

sos run ~/bioworkflows/variant-calling/gatk_joint_calling.ipynb vcf_qc
--container-option /mnt/vast/hpc/csg/containers/gatk4-annovar.sif
--vcf-prefix /mnt/vast/hpc/csg/Thashi/Wave4_AFRbatch3_2/AFRBatch3_2_Joint_calling/Wave4_AfrBatch3_2
--cwd /mnt/vast/hpc/csg/Thashi/Wave4_AFRbatch3_2/AFRBatch3_2_Joint_calling/
--vcf-filter basic