Set the path to your reference file with the NCM_REF environment variable eg. export NCM_REF=/// 1.14 samtools mpileup -uf $NCM_REF -l /usr/local/NGSCheckMate/SNP/SNP_GRCh38_hg38_wChr.bed aih-tih-sc-176637-R1_B23WHYVLT4_1.Aligned.sortedByCoord.out.bam | bcftools call -c > ./aih-tih-sc-176637-R1_B23WHYVLT4_1.Aligned.sortedByCoord.out.vcf Generate Data Set from . using this bed file : /usr/local/NGSCheckMate/SNP/SNP_GRCh38_hg38_wChr.bed