D3b RADIANT Annotation

Workflow to add various transcript and site level annotations to a DNA germline VCF. The workflow has the following framework:

• Old annotations are stripped with bcftools
• VCF is annotated with bcftools
• VCF is normalized
• VCF is annotated with VEP
• VCF is annotated with Echtvar
• Slivar Compound Hets is run
• Exomiser is run

Disabling Tools

Any of the tools can be enabled/disabled using the following disable_(insert_step) parameters. The default workflow runs in the following configuration:

• disable_bcftools_strip_anno = false
• disable_bcftools_norm = false
• disable_vep = false
• disable_echtvar_anno = false
• disable_slivar_compound_hets = true
• disable_exomiser = true

While any step can be disabled, it might have adverse affects on downstream tools. For example, if you disable VEP but turn on Slivar compound hets, the program can fail if your input VCF does not have any ANN or CSQ INFO fields. Validation safeguards only exist for each step because we want to allow for scenarios where partially annotated VCFs can enter at any stage. This allows for faster reannotation in the future.

Inputs

Required - Need to provide

As mentioned above requirements depend on what is enabled. At the minimum, three inputs are required.

• vcf: KF VCF to process
• vcf_index = Index of vcf
• output_basename: String prefix of file outputs. Technically optional, but strongly recommended. Default is TEST

BCFTOOLS STRIP ENABLED

If you enable BCFTOOLS STRIP, at least one of the following must be provided:

• rm_fields_csv
• annotate_vcf
• bcftools_strip_extra_args

BCFTOOLS NORM ENABLED

If you enable BCFTOOLS NORM, all of the following must be provided:

• fasta

VEP ENABLED

If you enable VEP, all of the following must be provided:

• assembly
• fasta
• vep_cache
• vep_cache_version
• vep_species

ECHTVAR ENABLED

If you enable ECHTVAR, all of the following must be provided:

• echtvar_zips

SLIVAR ENABLED

If you enable SLIVAR, all of the following must be provided:

• ped

As mentioned above, Slivar does require some annotations. For more information see our notes.

EXOMISER ENABLED

• analysis_file: YAML with analysis options. See https://exomiser.readthedocs.io/en/latest/advanced_analysis.html#analysis. Recommend default_exomiser_WGS_analysis.yml

• datadir_file: TAR GZ with reference files. Example contents:

  data/
  data/remm/
  data/remm/ReMM.v0.4.hg38.tsv.gz.tbi
  data/remm/ReMM.v0.4.hg38.tsv.gz
  data/2406_hg38.sha256
  data/2406_hg38/
  data/2406_hg38/2406_hg38_transcripts_refseq.ser
  data/2406_hg38/2406_hg38_transcripts_ensembl.ser
  data/2406_hg38/2406_hg38_genome.mv.db
  data/2406_hg38/2406_hg38_variants.mv.db
  data/2406_hg38/2406_hg38_transcripts_ucsc.ser
  data/2406_hg38/2406_hg38_clinvar.mv.db
  data/cadd/
  data/cadd/1.7/
  data/cadd/1.7/whole_genome_SNVs.tsv.gz
  data/cadd/1.7/whole_genome_SNVs.tsv.gz.tbi
  data/cadd/1.7/gnomad.genomes.r4.0.indel.tsv.gz
  data/cadd/1.7/gnomad.genomes.r4.0.indel.tsv.gz.tbi
  data/2406_phenotype/
  data/2406_phenotype/2406_phenotype.mv.db
  data/2406_phenotype/rw_string_10.mv
  data/2406_phenotype/phenix/
  data/2406_phenotype/phenix/out/
  data/2406_phenotype/phenix/out/10.out
  data/2406_phenotype/phenix/out/9_symmetric.out
  data/2406_phenotype/phenix/out/14.out
  data/2406_phenotype/phenix/out/13_symmetric.out
  data/2406_phenotype/phenix/out/20.out
  data/2406_phenotype/phenix/out/19.out
  data/2406_phenotype/phenix/out/16_symmetric.out
  data/2406_phenotype/phenix/out/7.out
  data/2406_phenotype/phenix/out/3_symmetric.out
  data/2406_phenotype/phenix/out/8.out
  data/2406_phenotype/phenix/out/17_symmetric.out
  data/2406_phenotype/phenix/out/15_symmetric.out
  data/2406_phenotype/phenix/out/5_symmetric.out
  data/2406_phenotype/phenix/out/11.out
  data/2406_phenotype/phenix/out/14_symmetric.out
  data/2406_phenotype/phenix/out/8_symmetric.out
  data/2406_phenotype/phenix/out/10_symmetric.out
  data/2406_phenotype/phenix/out/1.out
  data/2406_phenotype/phenix/out/20_symmetric.out
  data/2406_phenotype/phenix/out/11_symmetric.out
  data/2406_phenotype/phenix/out/18_symmetric.out
  data/2406_phenotype/phenix/out/4.out
  data/2406_phenotype/phenix/out/19_symmetric.out
  data/2406_phenotype/phenix/out/2.out
  data/2406_phenotype/phenix/out/12.out
  data/2406_phenotype/phenix/out/15.out
  data/2406_phenotype/phenix/out/2_symmetric.out
  data/2406_phenotype/phenix/out/9.out
  data/2406_phenotype/phenix/out/17.out
  data/2406_phenotype/phenix/out/16.out
  data/2406_phenotype/phenix/out/6_symmetric.out
  data/2406_phenotype/phenix/out/13.out
  data/2406_phenotype/phenix/out/18.out
  data/2406_phenotype/phenix/out/3.out
  data/2406_phenotype/phenix/out/4_symmetric.out
  data/2406_phenotype/phenix/out/1_symmetric.out
  data/2406_phenotype/phenix/out/7_symmetric.out
  data/2406_phenotype/phenix/out/6.out
  data/2406_phenotype/phenix/out/5.out
  data/2406_phenotype/phenix/out/12_symmetric.out
  data/2406_phenotype/phenix/hp.obo
  data/2406_phenotype/phenix/ALL_SOURCES_ALL_FREQUENCIES_genes_to_phenotype.txt
  data/2406_phenotype/hp.obo
  

• exomiser_pheno_file: YAML file with sample information

  • Individual example:

  ---
  id: FM_ZCF17CPC
  proband:
  subject:
      id: BS_4JPDCXVR
      sex: MALE
  phenotypicFeatures:
      - type:
          id: HP:0009733
          label: Glioma
  pedigree:
  persons:
      - individualId: BS_4JPDCXVR
      sex: MALE
      affectedStatus: AFFECTED
  
  metaData:
  resources:
      - id: hp
      name: human phenotype ontology
      url: http://purl.obolibrary.org/obo/hp.owl
      version: hp/releases/2019-11-08
      namespacePrefix: HP
      iriPrefix: 'http://purl.obolibrary.org/obo/HP_'
  phenopacketSchemaVersion: 2.0

  • Family example:

  ---
  id: FM_ASMHHE2N
  proband:
  subject:
      id: BS_KPAR4N94
      sex: MALE
  phenotypicFeatures:
      - type:
          id: HP:0009733
  pedigree:
  persons:
      - individualId: BS_KPAR4N94
      sex: MALE
      paternalId: BS_264QQD38
      maternalId: BS_EQ98CJJT
      affectedStatus: AFFECTED
      - individualId: BS_264QQD38
      sex: MALE
      affectedStatus: UNAFFECTED
      - individualId: BS_EQ98CJJT
      sex: FEMALE
      affectedStatus: UNAFFECTED
  metaData:
  resources:
      - id: hp
      name: human phenotype ontology
      url: http://purl.obolibrary.org/obo/hp.owl
      version: hp/releases/2019-11-08
      namespacePrefix: HP
      iriPrefix: 'http://purl.obolibrary.org/obo/HP_'
  phenopacketSchemaVersion: 2.0

Input Defaults and Run Configurations

All of the defaults can be found in params block of nextflow.config. Kids First specific run configurations can be found in conf/kids_first.config.

Kids First Run Configuration

Here's a snapshot of the run configuration for Kids First Germline data:

• bcftools strip uses the annotate module to:

  • remove existing VEP (CSQ) and gnomad 3.1.1 (gnomad_3_1_1*) annotations
  • add annotations from annotate_vcf

• bcftools norm: Uses norm to split (or join) mulit-allelics and normalize indels

• VEP uses cache 105 and meets the following specific criteria:

  --af_1kg
  --af_esp
  --af_gnomad
  --allele_number
  --allow_non_variant
  --appris
  --assembly GRCh38
  --buffer_size 100000
  --cache
  --cache_version 105
  --canonical
  --ccds
  --check_existing
  --compress_output bgzip
  --dir_cache ./
  --domains
  --dont_skip
  --failed 1
  --fasta Homo_sapiens_assembly38.fasta
  --flag_pick
  --format vcf
  --gene_phenotype
  --hgvs
  --hgvsg
  --mane
  --merged
  --no_escape
  --no_stats
  --numbers
  --offline
  --pick_order rank,biotype,mane,canonical,appris,tsl,ccds,length,ensembl,refseq
  --polyphen b
  --protein
  --pubmed
  --regulatory
  --shift_hgvs 1
  --sift b
  --species homo_sapiens
  --symbol
  --total_length
  --tsl
  --uniprot
  --variant_class
  --vcf
  --xref_refseq
  

• echtvar: Used for fast gnomAD annotation from the echtvar_zips input (gnomad.v3.1.1.custom.echtvar.zip)

• slivar: Used for rare variant discovery, primarily in joint family VCFs using the expr with the slivar_zips input (topmed.hg38.dbsnp.151.zip) and compount-hets modules

• exomiser: "The Exomiser is a Java program that finds potential disease-causing variants from whole-exome or whole-genome sequencing data." Requires phenotype information. See https://github.com/exomiser/Exomiser for more details

• annot_vcf_infix: For ANNOTATED_VCF output files only, give a string here to override the common string after output basename and before .vcf.gz Normally the output name is params.output_basename + tool-contributed string + vcf.gz[.tbi] which becomes: params.output_basename + params.annot_vcf_infix + vcf.gz[.tbi]

• min_disk: Currently CAVATICA-specific, set min disk size to save on EBS storage. Default 100GB (exomier tool min 400)

Bix Ops Cheatsheet

See here