VAFator 1.0.0

Annotate a VCF file with AF, AD and DP from tumor and normal BAMs

VAFator

VAFator annotates the variants in a VCF file with technical annotations from multiple BAM files. Supports annotating somatic variant calls with the annotations from the normal and the tumor samples; although it can also be used for germline variant calls.

Annotations:

• Allele frequency (AF): ratio of reads supporting the alternate allele.
• Allele count (AC): count of reads supporting the alternate allele.
• Depth of coverage (DP): number of reads covering the position of the variant

Outputs a VCF with the following annotations in the INFO field for tumor and normal:

chr1    12345       .       A       G       .       PASS  tumor_af=0.0;tumor_ac=0;tumor_dp=89;normal_af=0.0196;normal_ac=1;normal_dp=51
chr2    12345       .       A       G,T       .       PASS  tumor_af=0.2,0.2;tumor_ac=2,2;tumor_dp=10;normal_af=0.0,0.0;normal_ac=0,0;normal_dp=10

NOTE: notice that VAFator does not annotate samples in the FORMAT field

How to install

Install from PyPI (pip install vafator) or from bioconda (conda install bioconda::vafator).

How to run

Run as follows:

vafator --input-vcf /path/yo/your.vcf \
--output-vcf /path/yo/your_vafator.vcf \ 
--normal-bams /path/to/your_normal.bam \
--tumor-bams /path/to/your_primary_tumor.bam,/path/to/your_metastasis_tumor.bam

Both tumor and normal BAMs are optional, it can annotate only with the tumor BAMs or with the normal BAMs.

If more than one BAM is provided for either the tumor or the normal then the annotations are calculated across all BAMs and for also each of them separately (eg: tumor_af provides the allele frequency across all tumor BAMs, tumor_af_1 and tumor_af_2 provide the allele frequency on the first and second BAM respectively).

Also, a --prefix parameter can be used to run VAFator multiple times over the same VCF without overwriting its annotations. For instance, first vafator [...] --prefix primary --tumor-bams /path/to/your_primary_tumor.bam and then vafator [...] --prefix metastasis --tumor-bams /path/to/your_metastasis_tumor.bam.

Use the parameters --mapping-quality and --base-call-quality to define the minimum quality values for each read. All reads with quality values velow these thresholds will be filtered out.

Overlapping reads from read pairs are not double counted. The read with the highest base call quality is chosen.

Filter for multi-allelic variants

Multi-allelic variants are those that have more than one alternative allele (e.g.: A>C,G). This tool allows to select the allele with the highest allele frequency and filter out the lower frequency allele.

Run as follows:

multiallelics-filter --input-vcf /path/to/your_vafator.vcf \
--output-vcf /path/to/your_vafator_filtered.vcf \
--tumor-sample-name <SAMPLE> 

The above will look for the annotation <SAMPLE>_af and for multi-allelic variants it will filter out those with lower frequencies. Beware, that if the multi-allelic variants are split into more than one line in the VCF nothing will be filtered out.

Run as a Nextflow pipeline

VAFator is available as a Nextflow pipeline for convenience.

Run as follows:

nextflow run tron-bioinformatics/vafator -r 1.0.0 -profile conda --input_files /path/to/your.tsv

where --input_files expects four tab-separated columns without a header:

Sample name	VCF	Tumor BAMs	Normal BAMs
sample_1	/path/to/sample_1.vcf	/path/to/sample_1_tumor_1.bam,/path/to/sample_1_tumor_2.bam	/path/to/sample_1_normal.bam
sample_2	/path/to/sample_2.vcf	/path/to/sample_2_tumor.bam	/path/to/sample_1_normal.bam

Optional parameters:

• --output: the folder where to publish output
• --skip_multiallelic_filter: skip the filtering of multiallelics by frequency in the tumor (only highest frequency variant at the same position is kept)
• --base_call_quality: minimum base call quality, reads with values below will be filtered out (default: 30)
• --mapping_quality: minimum mapping quality, reads with values below will be filtered out (default: 1)
• --prefix: when provided the annotations are preceded by this prefix (e.g.: _tumor_ac, _tumor_af, etc), otherwise the annotations are named as tumor_af, normal_af, tumor_ac, normal_ac, tumor_dp and normal_d

Support for indels

VAFator provides equivalent annotations for indels. Depth of coverage and allele frequency are calculated on the position immediately before the indel. Only insertions and deletions as recorded in the CIGAR matching the respective coordinates and sequence from the VCF file are taken into account. Any read supporting a similar but not equal indel will not be counted. Also, multiallelic mutations are not supported for indels.

Support for MNVs

Not supported