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SAMPLE_ANALYSIS_README.md

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KFDRC PacBio HiFi WGS Variant Workflow

The KFDRC PacBio HiFi WGS Variant Workflow performs read alignment, variant calling, and phasing. This CWL is a conversion of PacBio's HiFi-human-WGS-WDL sample_analysis.wdl.

Relevant Softwares and Versions

Inputs

  • Universal Recommended:

    • bam: Unaligned sample BAM.
    • reference_fasta: Reference genome and index.
    • sample_id: Used to name outputs.

  • HiFiCNV

    • exclude_bed: Compressed BED and index of regions to exclude from calling by HiFiCNV (recommended: cnv.excluded_regions.common_50.hg38.bed.gz).
    • expected_bed_female: BED of expected copy number for female karyotype for HiFiCNV (recommended: expected_cn.hg38.XX.bed).
    • expected_bed_male: BED of expected copy number for male karyotype for HiFiCNV (recommended: expected_cn.hg38.XY.bed).

  • Tandem Repeat

    • Recommended:
      • reference_tandem_repeat_bed: Tandem repeat locations used by pbsv to normalize SV representation (recommended: human_GRCh38_no_alt_analysis_set.trf.bed).
      • trgt_tandem_repeat_bed: Tandem repeat sites to be genotyped by TRGT (recommended: human_GRCh38_no_alt_analysis_set.trgt.v0.3.4.bed).
    • Optional:
      • sex: ["MALE", "FEMALE", null]. If the sex field is missing or null, sex will be set to unknown. Used to set the expected sex chromosome karyotype for TRGT and HiFiCNV (defaults to karyotype XX).

  • DeepVariant

    • Recommended:
      • model: TensorFlow model checkpoint to use to evaluate candidate variant calls. Default is set to PACBIO for PacBio data.
    • Optional:
      • custom_model: Alternatively, a custom TensorFlow model checkpoint may be used to evaluate candidate variant calls. If not provided, the model trained by the DeepVariant team will be used.

A reference data bundle for this pipeline can be found here.

# download the reference data bundle
wget https://zenodo.org/records/8415406/files/wdl-humanwgs.v1.0.2.resource.tgz?download=1

# extract the reference data bundle and rename as dataset
tar -xzf wdl-humanwgs.v1.0.2.resource.tgz && mv static_resources PacBio_reference_bundle

Outputs

  • BAM stats and alignment

    • bam_stats: TSV of length and quality for each read.
    • read_length_summary: Read length distribution.
    • read_quality_summary: Read quality distribution.
    • aligned_bam: Aligned BAM.
    • svsig: Structural variant signatures.

  • Small variants

    • deepvariant_vcf: Small variants (SNPs and INDELs < 50bp) VCF called by DeepVariant (with index).
    • deepvariant_gvcf: Small variants (SNPs and INDELs < 50bp) gVCF called by DeepVariant (with index).
    • deepvariant_vcf_stats: bcftools stats summary statistics for small variants.
    • deepvariant_roh_out: Output of bcftools roh using --AF-dflt 0.4.
    • deepvariant_roh_bed: Regions of homozygosity determiend by bcftools roh using --AF-dflt 0.4.

  • Structural variants

    • pbsv_call_vcf: Structural variants called by pbsv (with index).

  • Phased variant calls and haplotagged alignments

    • phased_deepvariant_vcf: Small variants called by DeepVariant and phased by HiPhase (with index).
    • phased_pbsv_vcf: Structural variants called by pbsv and phased by HiPhase (with index).
    • phased_summary: Phasing summary TSV file.
    • hiphase_stats: Phase block summary statistics written by HiPhase.
    • hiphase_blocks: Phase block list written by HiPhase.
    • hiphase_haplotags: Per-read haplotag information, written by HiPhase.
    • hiphase_bam: Aligned (by pbmm2), haplotagged (by HiPhase) reads (with index).
    • haplotagged_bam_mosdepth_summary: mosdepth summary of median depths per chromosome.
    • haplotagged_bam_mosdepth_region_bed: mosdepth BED of median coverage depth per 500 bp window.
    • paraphase_output_json: Paraphase summary file.
    • paraphase_realigned_bam: Realigned BAM for selected medically relevant genes in segmental duplications (with index).
    • paraphase_vcfs: Phased Variant calls for selected medically relevant genes in segmental duplications.

  • Tandem repeat information

    • trgt_spanning_reads: Fragments of HiFi reads spanning loci genotyped by TRGT (with index).
    • trgt_repeat_vcf: Tandem repeat genotypes from TRGT (with index).

  • Methylation

    • cpg_pileup_beds: 5mCpG site methylation probability pileups.
    • cpg_pileup_bigwigs: 5mCpG site methylation probability pileups.

  • CNVs

    • hificnv_vcf: VCF output containing copy number variant calls for the sample from HiFiCNV.
    • hificnv_copynum_bedgraph: Copy number values calculated for each region.
    • hificnv_depth_bw: Bigwig file containing the depth measurements from HiFiCNV.
    • hificnv_maf_bw: Bigwig file containing the minor allele frequency measurements from DeepVariant, generated by HiFiCNV.

Estimated Run Times

We processed a 26.5 GB BAM file using the KFDRC PacBio HiFi WGS Variant Workflow with default settings on CAVATICA. Here are the details of the run:

  • Run Time: 12 hours, 49 minutes
  • Cost: $10.22

Other Resources