BCFtools tutorial | mpileup | variant calling

BCFtools is a set of utilities that manipulate variant calls in the Variant Call Format (VCF) and its binary counterpart BCF. All commands work transparently with both VCFs and BCFs, both uncompressed and BGZF-compressed.

Note : Spelling mistake on "previous video"

1️⃣ Check the mpileup usage

./bcftools mpileup

2️⃣ BAM to BCF conversion

./bcftools mpileup --threads 4 -E -O b -f Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa AS_W_ETOH.bam | ./bcftools call --threads 4 -mv -O b -o AS_W_ETOH_calls.bcf

./bcftools mpileup --threads 4 -E -O b -f Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa AS_WO_ETOH.bam | ./bcftools call --threads 4 -mv -O b -o AS_WO_ETOH_calls.bcf

####### mpileup .. multi-way pileup producing genotype likelihoods
####### --threads INT Use multithreading with INT worker threads [0]
####### -E, --redo-BAQ Recalculate BAQ on the fly, ignore existing BQs
####### -O, --output-type TYPE 'b' compressed BCF; 'u' uncompressed BCF;
'z' compressed VCF; 'v' uncompressed VCF; 0-9 compression level [v]

####### -d, --max-depth INT Max raw per-file depth; avoids excessive memory usage [250]
####### -f, --fasta-ref FILE Faidx indexed reference sequence file

Base Alignment Quality (BAQ):
BAQ is a statistical method developed to refine the quality scores (Phred scores) associated with each base in a read alignment

####### call .. SNP/indel calling (former "view")
####### -m, --multiallelic-caller Alternative model for multiallelic and rare-variant calling (conflicts with -c)
####### -v, --variants-only Output variant sites only
####### -O, --output-type b|u|z|v Output type: 'b' compressed BCF; 'u' uncompressed BCF; 'z' compressed VCF; 'v' uncompressed VCF [v]
####### -o, --output FILE Write output to a file [standard output]


3️⃣ BCF to VCF conversion

./bcftools view AS_W_ETOH_calls.bcf % AS_W_ETOH_calls.vcf

./bcftools view AS_WO_ETOH_calls.bcf % AS_WO_ETOH_calls.vcf

replace the % with angular backet

4️⃣ Check on Libre Calc




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====================== Follow genomic variants analysis pipeline =====================
------ Tutorial made in the year 2024-2025

🎯Step 1: Download fastq(raw file-SRA) file from NCBI
   • How to download fastq file | Full tutorial...  

🎯Step 2: Fastq file quality check [Fastqc tool]
   • How to install fastqc in windows | Beginne...  

🎯Step 3: Trim the adapter and bad reads from the fastq file [Fastp tool]
   • Fastp | Install | Run | Paired end #bioinf...  

🎯Step 4: Mapping/Alignment of fastq to generate SAM file [STAR tool]
   • STAR | Install | Build index | #bioinforma...  
   • STAR | sequence alignment | mapping  #bioi...  

🎯Step 5: Sort the SAM file into BAM [ Samtools ]
   • Samtools | Install | SAM to BAM conversion...  

🎯Step 6: AddOrReplaceReadGroup of BAM files [GATK tools]
   • GATK Installation  | AddOrReplaceReadGroup...  

🎯Step 7: Mark Duplication of BAM files [GATK tools]
   • GATK Tutorial  | Mark Duplication | Varian...  

🎯Step 8: Merge BAM files [ Samtools ]
   • BCFtools installation | Merge BAM files #b...  

🎯Step 9: mpileup for variant calling [ BCFtools ]
   • BCFtools tutorial | mpileup | variant call...  

🎯Step 10: snp analysis [snpEFF tools]
   • snpEff tutorial | Installation | variant c...  

====================== Subscribe to my channel, and learn complete genomics analysis
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=========== Jump to
00:05 Introduction
00:25 Add or Replace read group
00:27 Mark duplicates
00:30 Marge bam files
00:35 Complete Genomic variant analysis pipeline
00:47 mpileup: Bam to BCF
07:50 BCF to VCF


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