Wakhan

Note: This repository is under extensive updates.

A tool to analyze haplotype-specific chromosome-scale somatic copy number aberrations and aneuploidy using long reads (Oxford Nanopore, PacBio). Wakhan takes long-read alignment and phased heterozygous variants as input, and first uses extends the phased blocks, taking advantage of the CNA differences between the haplotypes. Wakhan then generates inetractive haplotype-specific coverage plots.

Breakpoints/SVs based segmentation and Copy numbers estimation:

Installation (enabling through conda environment)

git clone https://github.com/KolmogorovLab/Wakhan.git
cd Wakhan/
conda env create -f environment.yml -n Wakhan
conda activate Wakhan
cd src/

Usage

Tumor-Normal Mode (requires normal phased VCF)

python main.py --threads <4> --reference <ref.fa>  --target-bam <data.tumor.bam>  --normal-phased-vcf <data.normal_phased.vcf.gz>  --genome-name <cellline/dataset name> --cut-threshold <150> --out-dir-plots <genome_abc_output> --breakpoints <severus-sv-VCF>

Tumor-Normal mixture and purity/ploidy estimation

User can input both --tumor-ploidy and --tumor-purity to inform copy number model about normal contamination in tumor to estimate copy number states correctly.

Tumor-only (requires tumor phased/haplotagged BAM and phased VCF)

python main.py --threads <4> --reference <ref.fa>  --target-bam <data.tumor_haplotagged.bam>  --tumor-vcf <data.tumor_phased.vcf.gz> --genome-name <cellline/dataset name> --cut-threshold <150> --out-dir-plots <genome_abc_output> --breakpoints <severus-sv-VCF>

Examples

Few cell lines arbitrary phase-switch correction and copy number estimation output with coverage profile is included in the examples directory.

Required parameters

--reference Reference file path
--target-bam path to target bam files (must be indexed)
--out-dir-plots path to output coverage plots
--genome-name genome cellline/sample name to be displayed on plots
--normal-phased-vcf normal phased VCF file to generate het SNPs frequncies pileup for tumor BAM (if tumor-only mode, use phased --tumor-vcf instead)
--tumor-vcf VCF file to plot snps frequencies, ratios and LOH regions (Note: phased VCF is required in tumor-only mode)
--breakpoints For segmentation to use in CN estimation, structural variations/breakpoints VCF is required

Optional parameters

--phaseblock-flipping-enable enabling phaseblock flipping in coverage plots
--phaseblocks-enable enabling phaseblocks display in coverage plots
--contigs List of contigs (chromosomes, default:chr1-22) to be included in the plots (Note: chrX, chrY support in CNA plots is not included yet) [e.g., chr1-22,X,Y]
--without-phasing enable it if CNA analysis is being performed without phasing

Output produced

<genome-name>_genome_copynumber.html Genome-wide copy number plots with coverage information on same axis
<genome-name>_copynumber_breakpoints.html Genome-wide copy number plots with coverage information on opposite axis, additionally breakpoints and genes annotations
bed_output It contains copy numbers and LOH (in case tumor VCF is provided) segments in bed format
coverage_plots Haplotype specific coverage plots for chromosomes with option for unphased coverage
variation_plots Copy number chromosomes-scale plots with segmentation, coverage and LOH/SNPs ratios (in case tumor VCF is provided)
phasing_output Phase-switch error correction plots and phase corrected VCF file (*rephased.vcf.gz)

Prerequisite

This tool requires haplotagged tumor BAM and phased VCF in case tumor-only mode and normal phased VCF in case tumor-normal mode. This can be done through any phasing tools like Margin, Whatshap and Longphase. Following commands could be helpful for phasing VCFs and haplotagging BAMs.

For normal/tumor pair:

# ClairS phase and haplotag both normal and tumor samples
singularity run clairs_latest.sif /opt/bin/run_clairs --threads 56 --phase_tumor True --use_whatshap_for_final_output_haplotagging --use_whatshap_for_final_output_phasing --tumor_bam_fn normal.bam --normal_bam_fn tumor.bam --ref ref.fasta --output_dir clairS --platform ont_r10

# Phase normal sample
pepper_margin_deepvariant call_variant -b normal.bam -f ref.fasta -o pepper/output -t 56 --ont_r9_guppy5_sup -p pepper --phased_output

# Haplotag tumor sample with normal phased VCF (phased.vcf.gz) output from previous step
whatshap haplotag --ignore-read-groups phased.vcf.gz tumor.bam  --reference ref.fasta -o tumor_whatshap_haplotagged.bam

For tumor only:

# Phase and haplotag tumor sample
singularity run clair3_latest.sif /opt/bin/run_clair3.sh --use_whatshap_for_final_output_haplotagging --use_whatshap_for_final_output_phasing --bam_fn=tumor.bam --ref_fn=ref.fasta --threads=56 --platform=ont --model_path=r941_prom_sup_g5014 --output=clair3 --enable_phasing

# Phase and haplotag tumor sample
pepper_margin_deepvariant call_variant -b tumor.bam -f ref.fasta -o pepper/output -t 56 --ont_r9_guppy5_sup -p pepper --phased_output

KolmogorovLab / Wakhan