Pathogenicity Preprocessing Workflow

To run, please see the CAVATICA app. Each version should correspond with a git release. This repo makes use of the git submodule feature for ease of code maintenance. To properly retrieve all relevant code:

git clone  https://github.com/d3b-center/D3b-Pathogenicity-Preprocessing
git submodule init
git submodule update

Prequisite

It is recommended to have first run the Kids First Germline Annotation Workflow.

Pathogenicity Preprocessing Workflow

This workflow uses the prerequisite input to run the InterVar workflow and autoPVS1 tool. The major pieces of software being used are:

• ANNOVAR latest: The software has no versioning, but references do. See annovar_db section in Recommended inputs
• InterVar v2.2.1
• AutoPVS1 v2.0.0: Modified from AutoPVS1 v2.0 to fit annotated KF vcf output. See README for autoPVS1 for details

Optionally, if you wish to add and, if needed, overwrite another annotation from a VCF file (likely ClinVar), a BCFtools strip and annotate steps are provided. The input VCF will be processed, and its result will appear as an additional output in the workflow.

Recommended inputs:

• annovar_db: ANNOVAR Database with at minimum required resources to InterVar. Need to use ANNOVAR download commands to get the following:

     annovar_humandb_hg38_intervar/
     ├── hg38_AFR.sites.2015_08.txt
     ├── hg38_AFR.sites.2015_08.txt.idx
     ├── hg38_ALL.sites.2015_08.txt
     ├── hg38_ALL.sites.2015_08.txt.idx
     ├── hg38_AMR.sites.2015_08.txt
     ├── hg38_AMR.sites.2015_08.txt.idx
     ├── hg38_EAS.sites.2015_08.txt
     ├── hg38_EAS.sites.2015_08.txt.idx
     ├── hg38_EUR.sites.2015_08.txt
     ├── hg38_EUR.sites.2015_08.txt.idx
     ├── hg38_SAS.sites.2015_08.txt
     ├── hg38_SAS.sites.2015_08.txt.idx
     ├── hg38_avsnp147.txt
     ├── hg38_avsnp147.txt.idx
     ├── hg38_clinvar_20210501.txt
     ├── hg38_clinvar_20210501.txt.idx
     ├── hg38_dbnsfp42a.txt
     ├── hg38_dbnsfp42a.txt.idx
     ├── hg38_dbscsnv11.txt
     ├── hg38_dbscsnv11.txt.idx
     ├── hg38_ensGene.txt
     ├── hg38_ensGeneMrna.fa
     ├── hg38_esp6500siv2_all.txt
     ├── hg38_esp6500siv2_all.txt.idx
     ├── hg38_gnomad_genome.txt
     ├── hg38_gnomad_genome.txt.idx
     ├── hg38_kgXref.txt
     ├── hg38_knownGene.txt
     ├── hg38_knownGeneMrna.fa
     ├── hg38_refGene.txt
     ├── hg38_refGeneMrna.fa
     ├── hg38_refGeneVersion.txt
     ├── hg38_rmsk.txt
     └── hg38_seq
         ├── annovar_downdb.log
         └── hg38.fa
  

• intervar_db: InterVar Database from git repo + mim_genes.txt
• autopvs1_db: git repo files plus a user-provided fasta reference. For hg38, recommend:

  data/
      ├── Homo_sapiens_assembly38.fasta
      ├── Homo_sapiens_assembly38.fasta.fai
      ├── PVS1.level
      ├── clinvar_pathogenic_GRCh38.vcf
      ├── clinvar_trans_stats.tsv
      ├── exon_lof_popmax_hg38.bed
      ├── expert_curated_domains_hg38.bed
      ├── functional_domains_hg38.bed
      ├── hgnc.symbol.previous.tsv
      ├── mutational_hotspots_hg38.bed
      └── ncbiRefSeq_hg38.gpe
  

• annovar_db_str: Name of dir created when annovar_db tar ball in decompressed. Default: annovar_humandb_hg38_intervar
• autopvs1_db_str: Name of dir created when autopvs1_db tar ball in decompressed. Default: data
• intervar_db_str: Name of dir created when intervar_db_str tar ball in decompressed. Default: intervardb

Note: We used a gene symbol liftover tool to allow gene symbols searches from different gene models to be found, PVS1.level was augmented with additional entries in which a gene symbols from the original file has changed.

The update_gene_symbols.py tool was used to achieve this, with liftover source obtained from here to match gene symbols from default/recommended VEP annotation. Example command:

python3 /Users/brownm28/Documents/git_repos/D3b-DGD-Collaboration/scripts/update_gene_symbols.py -g hgnc_complete_set_2021-06-01.txt -f PVS1.level -z GENE level -u GENE -o results --explode_records 2> old_new.log

With results used to replace PVS1.level file. Recommend references for this workflow can be obtained here.

InterVar Classification Workflow

This workflow is a critical component in generating scoring metrics needed to classify pathogenicity of variants. Documentation for this can be found here

AutoPVS1

An additional pathogenicity scoring tool, run on the VEP-annotated input. Documentation for this can be found here

Optional Inputs

As mentioned above, the preprocessing workflow can add an additional annotation

• annotation_vcf: hg38 chromosome-formatted VCF. If provided BCFtools will add annotation from the specified columns for each variant that matches
• bcftools_annot_columns: A CSV string of from annotation to port into the input vcf. Must provide if annotation_vcf given. See BCFtools annotate documentation on how to properly reference
• bcftools_strip_for_vep: If re-annotating certain INFO fields, it's best to strip the old annotation first to avoid conflicts. Use the same format as bcftools_annot_columns to reference fields being stripped
• bcftools_strip_for_annovar: More of a convenience to strip the ANNOVAR VCF of annotations that maybe have been used initially in the workflow, but will likely not be used downstream

A note on ClinVar annotation

For the publication, ClinVar release 20231028 was used. In order to be compatible with our hg38-aligned VCFs, we additionally downloaded the variant summary file, ran a custom script that:

• Converted contigs to chr format
• Dropped contigs not in hg38
• Use the variant summary table to replace N alleles and split into canonical ACGT alleles as those N were actually representing extended IUPAC nucleotides

Command run:

scripts/cleanup_clinvar.py --input_vcf clinvar_20231028.vcf.gz --variant_summary variant_summary.txt.gz --update_json docs/update_clinvar.json --output_filename clinvar_20231028.hg38_fmt.vcf.gz --threads 4