Contact: Stephen Montgomery (smontgom@stanford.edu), Rachel Ungar (raungar@stanford.edu), Page Goddard (pgoddard@stanford.edu), Tanner Jensen (tannerj@stanford.edu) This pipeline is an iteration on the pipeline released in the paper Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts

Pipeline Overview

Under scripts

Analysis Overview

Under analysis_scripts

Installation Preparation

• if necessary, install Miniconda as recommended here

  wget https://docs.conda.io/en/latest/miniconda.html#linux-installers
  bash Miniconda3-latest-Linux-x86_64.sh
  

• install snakemake via conda and mamba as recommended here

n conda install -c conda-forge mamba mamba create -c conda-forge -c bioconda -n snakemake snakemake conda activate snakemake snakemake --help

• Set up a snakemake slurm profile (good install walkthrough here) and information on actual use here

  • Setting up the slurm profile requires cookiecutter, which can be installed with conda:

  conda install -c conda-forge cookiecutter
  

environment_paths.yaml

There should be no direct paths in the snakemake. Please add any paths to the yaml file.

External Data Resources

Utilities

• bigBedToBed downloaded from: https://hgdownload.soe.ucsc.edu/admin/exe/
• GENCODE backmap: https://github.com/diekhans/gencode-backmap

Genome Assemblies

• hg19: GRCh37.primary_assembly.genome.fa.gz
• hg38: GRCh38.primary_assembly.genome.fa.gz
• CHM13v2.0: CHM13v2.0_maskedY.fa.gz

Genome Annotations

• hg19 annotation: https://www.gencodegenes.org/human/release_35lift37.html > Comprehensive gene annotation (Regions: CHR)
  • https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_35/GRCh37_mapping/gencode.v35lift37.annotation.gtf.gz
• hg38 annotation: https://www.gencodegenes.org/human/release_35.html > Comprehensive gene annotation (Regions: CHR)
  • https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_35/gencode.v35.annotation.gtf.gz
• CHM13v2.0 Annotation: https://github.com/marbl/CHM13#gene-annotation > UCSC GENCODEv35 CAT/Liftoffv2
  • https://s3-us-west-2.amazonaws.com/human-pangenomics/T2T/CHM13/assemblies/annotation/chm13.draft_v2.0.gene_annotation.gff3
• hg19 vs hg38 annotation backmap table: https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_35/GRCh37_mapping/gencode.v35lift37.map-info.tsv.gz

Blacklisted Regions & Known Issues

• ENCODE Blacklisted Regions for hg19 and hg38: https://github.com/Boyle-Lab/Blacklist/tree/master/lists
• CHM13 Blacklisted Regions: https://bedbase.org/#/bedsplash/8329d8c624880308ab51ba05149a737d
• Official GRC Issues and Incidents Database: https://www.ncbi.nlm.nih.gov/grc/human/issues
  • data scraped from https://www.ncbi.nlm.nih.gov/projects/genome/assembly/grc/human/issues/filter-data-django.js?d=20150303
  • UCSC Genome Browswer track and download for hg19 issue track from GRC: https://genome.ucsc.edu/cgi-bin/hgTables?db=hg19&hgta_group=map&hgta_track=grcIncidentDb&hgta_table=grcIncidentDb&hgta_doSchema=describe+table+schema
  • UCSC Genome Browswer track and download for hg38 issue track from GRC: https://genome.ucsc.edu/cgi-bin/hgTables?db=hg38&hgta_group=map&hgta_track=grcIncidentDb&hgta_table=grcIncidentDb&hgta_doSchema=describe+table+schema
• Official documentation of known issues in the CHM13 assembly: https://github.com/marbl/CHM13-issues/blob/main/v2.0_issues.bed

Build Changes

• hg19 vs hg38 build differences
  • hg38 coordinates: hg38ContigDiff.txt.gz: https://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/ Aug 1 2022
  • hg19 coordinates: hg19ContigDiff.txt.gz: https://hgdownload.cse.ucsc.edu/goldenpath/hg19/database/ Aug 1 2022
  • Table schema / track description: https://genome.ucsc.edu/cgi-bin/hgTables?db=hg38&hgta_group=map&hgta_track=hg38ContigDiff&hgta_table=hg38ContigDiff&hgta_doSchema=describe+table+schema
• CHM13 vs HG38 non-syntenic regions (downloaded Oct 19 2022): https://hgdownload.soe.ucsc.edu/hubs/GCA/009/914/755/GCA_009914755.4/bbi/GCA_009914755.4_T2T-CHM13v2.0.hgUnique/chm13v2-unique_to_hg38.bed
• Build differences in CHM13 vs hg38 for medically relevant genes from table S13: https://www.science.org/doi/10.1126/science.abl3533