Running ORE

Overview

Pre-processing data

Required inputs

1. Gene transcription start site (TSS) location with expression as Browser Extensible Data (BED)
2. Genotypes as Variant Call Format (VCF)

ORE uses the same inputs as FastQTL, bgzip with bcftools and index with tabix:

# Prepare the VCF
bgzip test.vcf
tabix -p vcf test.vcf.gz
# Prepare the expression BED file
bgzip test.bed
tabix -p bed test.bed.gz

Normalize the VCF using bcftools norm. Standardize every allele into its most parsimonious and left aligned form (so that appropriate allele frequencies can be obtained from population databases)

bcftools norm -f human_g1k_v37.fasta -Oz test.vcf.gz -o test.norm.vcf.gz

Download population databases

1. Register with ANNOVAR
2. You will get an email for downloading ANNOVAR. Download this and unzip/untar the file using the following commands (replacing USER_KEY with what was given in the email)

wget http://www.openbioinformatics.org/annovar/download/USER_KEY/annovar.latest.tar.gz
tar -xzvf annovar.latest.tar.gz

3. Download the databases (~20 Gb) with these commands

cd annovar
perl annotate_variation.pl -buildver hg19 -downdb -webfrom annovar refGene humandb/
perl annotate_variation.pl -buildver hg19 -downdb -webfrom annovar ensGene humandb/
perl annotate_variation.pl -buildver hg19 -downdb -webfrom annovar gnomad_genome humandb/

4. Set the database directory as an environmental variable

DB_DIR="./human_db/"

Run in Python virtual environment

Among many other benefits, running in a virtual environment allows one to install ORE without administrator privileges (useful for working in shared scientific computing environments).

# Create the virtual environment
virtualenv venv_ore
# Enter the environment
source venv_ore/bin/activate
# make sure that pip points to the virtual environment python directory by install the latest version
pip install --upgrade pip
# install ORE
pip install ore
# leave the virtual environment
deactivate
# re-enter the virtual environment
source venv_ore/bin/activate

Specify parameters

Required arguments:

-v VCF, --vcf VCF
	Location of VCF file. Must be tabixed!
-b BED, --bed BED
	Gene expression file location. Must be tabixed!

Optional file locations:

-o OUTPUT, --output OUTPUT
	Output prefix
--outlier_output OUTLIER_OUTPUT
	Outlier filename
--enrich_file ENRICH_FILE
	Output file for enrichment odds ratios and p-values

Optional outlier arguments:

--extrema	Only the most extreme value is an outlier
--distribution DISTRIBUTION
	Outlier distribution. Options: {normal,rank,custom}
--threshold THRESHOLD
	Expression threshold for defining outliers. Must be greater than 0 for normal or (0,0.5) non-inclusive with rank. Ignored with custom
--max_outliers_per_id MAX_OUTLIERS_PER_ID
	Maximum number of outliers per ID

Optional variant-related arguments:

--af_rare AF_RARE
	AF cut-off below which a variant is considered rare (space separated list e.g., 0.1 0.05)
--af_vcf	Use the VCF AF field to define an allele as rare.
--intracohort_rare_ac INTRACOHORT_RARE_AC
	Allele COUNT to be used instead of intra-cohort allele frequency. (still uses af_rare for population level AF cut-off)
--gq GQ	Minimum genotype quality each variant in each individual
--dp DP	Minimum depth per variant in each individual
--aar AAR	Alternate allelic ratio for heterozygous variants (provide two space-separated numbers between 0 and 1, e.g., 0.2 0.8)
--tss_dist TSS_DIST
	Variants within this distance of the TSS are considered
--upstream	Only variants UPstream of TSS
--downstream	Only variants DOWNstream of TSS

Optional arguments for using ANNOVAR:

--annovar	Use ANNOVAR to specify allele frequencies and functional class
--variant_class
	Only variants in these classes will be considered. Options: {intronic,intergenic,exonic,UTR5,UTR3,splicing,upstream,ncRNA}
--exon_class	Only variants with these exonic impacts will be considered. Options: {nonsynonymous,intergenic,nonframeshift,frameshift,stopgain,stoploss}
--refgene	Filter on RefGene function.
--ensgene	Filter on ENSEMBL function.
--annovar_dir ANNOVAR_DIR
	Directory of the table_annovar.pl script
--humandb_dir HUMANDB_DIR
	Directory of ANNOVAR data (refGene, ensGene, and gnomad_genome)

optional arguments:

-h, --help	show this help message and exit
--version	show program’s version number and exit
--processes PROCESSES
	Number of CPU processes
--clean_run	Delete temporary files from the previous run

Run

Run ORE using the desired parameters. Currently ORE creates many temporary files that allow for faster re-running or picking up in case of a run-time crash or error.