Kraf (Pronunce Carafe) - Kmer based Recalibration of Allele Frequency

Allele frequency estimation for long deletions made by variant caller (eg. freebayes) are usually underestimated due to the fact that these softwares only compare reads with identified breakpoint to the coverage at the position of the deletion. Also some read may also contained the deletion, but near their extremity, and the alignement of the deletion was not possible resulting in softclipping.

Method

Kraf uses a k-mers strategy to recalibrate the Allele frequency of these deletions using the signal observed on the k-mer level. This strategy is alignment-free and results for this variants into a better estimation of the AF.

Kraf process as follow, each deletion is being processed and converted in two set of k-mers :

• alt k-mers : that overlap the breakpoint and will help to capture reads with the deletion
• ref k-mers : that overlap the reference sequence that was deleted and will help to capture reads without a deletion

These k-mers are then quantified in the reads sequences from the BAM files. All reads are processed (even unmapped) except duplicates.

Finally we take the median of the alt k-mers counts and the median of the ref k-mers counts and we compute a refined VAF based on those values.

The refined VAF is only set if :

• it is superior the previous computed VAF (extracted from either AF or AD field)
• it is inferior to 1 (miscounting of the deletion k-mers could produce this behaviour)
• [desactivated] it comprised between the previous VAF and 4 times the previous VAF.

Usage

Kraf takes in input the reference genome in fasta format, a BAM file and the VCF with called variants. It output on STDOUT a modified VCF with updated VAF for deletions.

Usage: kraf [options] <fasta> <bam> <vcf>

Arguments:
    <fasta>           Reference genome in FASTA format.
    <bam>             BAM files with aligned reads
    <vcf>             VCF files with called variants

Options:
    -k <int>          K-mer size to use for recalibration. [default: 31]
    -p <int>          Padding size of k-mers around the deletion. [default: 5]
    -t <int>          Number of decompression threads. [default: 4]
    --min-del <int>   Minimun deletion size elligible for AF recalibration [default: 10]
    -v, --verbose     Enable verbose mode

KRAF will set the following values in the FORMAT field of the sample :

• AO : Number of alternate observations
• AD : Allelic depths for the ref and alt alleles in the order listed.
• AF : Allele Frequency

When an AF, AO and AD are beeing replaced in the FORMAT field a new TAG named "OLD_TAG" (ex: OLD_AF) is appended in order to keep the trace of the previous values computed by the caller and replaced by KRAF.

Beware of these Kraf limitations :

• Only works for 1 sample VCF file
• Only deals with deletions

Install

First install dependencies : nim compilator and hts-nim library

apt-get install nim
nimble install -y hts

Compile kraf binary and place it somewhere available from your $PATH:

git clone https://gitlab.seq.one/workset/kraf.git && cd kraf
make

TODO

• Recalibrate other variant than just deletions
• We could test the distribution of k-mer counts to see if there is somthing abdnormal beforme recalibration the VAF with the median value. Indeed if we have non specific k-mers targeting the variant or the reference we could over (or under) estimate the VAF
• A given k-mer should not be counted as both reference and alternative, it should be specific.