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Bcftools
2. Program guides

Bcftools

Bcftools are a set of utilities for variant calling and manipulating VCFs and BCFs.

Generating genotype likelihoods for alignment files using bcftools mpileup

bcftools mpileup can be used to generate VCF or BCF files containing genotype likelihoods for one or multiple alignment (BAM or CRAM) files as follows:

$ bcftools mpileup --max-depth 10000 --threads n -f reference.fasta -o genotype_likelihoods.bcf reference_sequence_alignmnet.bam

In this command...

  1. --max-depth or -d sets the reads per input file for each position in the alignment. In this case, it is set to 10000
  2. --threads sets the number (n) of processors/threads to use.
  3. --fasta-ref or -f is used to select the faidx-indexed FASTA nucleotide reference file (reference.fasta) used for the alignment.
  4. --output or -o is used to name the ouput file (genotype_likelihoods.bcf).
  5. The final argument given is the input BAM alignment file (reference_sequence_alignment.bam). Multiple input files can be given here.

Variant calling using bcftools call

bcftools call can be used to call SNP/indel variants from a BCF file as follows:

$ bcftools call -O b --threads n -vc --ploidy 1 -p 0.05 -o variants_unfiltered.bcf genotype_likelihoods.bcf

In this command...

  1. --output-type or -O is used to select the output format. In this case, b for BCF.
  2. --threads sets the number (n) of processors/threads to use.
  3. -vc specifies that we want the output to contain variants only, using the original SAMtools consensus caller.
  4. --ploidy specifies the ploidy of the assembly.
  5. --pval-threshold or -p is used to the set the p-value threshold for variant sites (0.05).
  6. --output or -o is used to name the ouput file (variants_unfiltered.bcf).
  7. The final argument is the input BCF file (genotype_likelihoods.bcf).

Filtering variants using bcftools filter

bcftools filter can be used to filter variants from a BCF file as follows...

$ bcftools filter --threads n -i '%QUAL>=20' -O v -o variants_filtered.vcf variants_unfiltered.bcf

In this command...

  1. --threads sets the number (n) of processors/threads to use.
  2. --include or -i is used to define the expression used to filter sites. In this case, %QUAL>=20 results in sites with a quality score greater than or equal to 20.
  3. --output-type or -O is used to select the output format. In this case, v for VCF.
  4. --output or -o is used to name the ouput file (variants_filtered.vcf).
  5. The final argument is the input BCF file (genotype_likelihoods.bcf).

See also

Futher reading