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08_Annotation.md

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Annotate locus-based analysis results

This guide provide an example of one method by which the ExpansionHunter Denovo case-control or outlier locus-based analysis results can be annotated with information about the gene and genomic region for each repeat locus identified.

A script to annotate ExpansionHunter Denovo locus-based results using ANNOVAR is provided here ExpansionHunterDenovo/scripts/annotate_ehdn.sh.

Note: Genomic regions reported by this annotation script need to be interpreted with caution.

Regions identified by ExpansionHunter Denovo are defined based on the locations of the supporting anchored in-repeat reads and do not exactly correspond to location of the repeat expansion itself. ANNOVAR denotes a region as 'exonic' if any part of the region overlaps with an exon.

A locus annotated as 'exonic' does not guarantee that the repeat expansion itself overlaps a coding region, only that the region defined by the supporting anchored in-repeats reads overlaps with an exon. However, it does suggest the expansion lies very close to a coding region.

Setup ANNOVAR

The provided annotation script requires an existing ANNOVAR installation and the refGene annotation database to be downloaded.

  1. Register and download ANNOVAR

  2. Unpack ANNOVAR in your home directory or any other directory in your PATH

    tar xvfz annovar.latest.tar.gz
mv annovar ~/annovar

  3. Download the refGene annotation database for the genome build you wish to use:

    perl ~/annovar/annotate_variation.pl -buildver hg19 -downdb -webfrom annovar refGene ~/annovar/humandb/
perl ~/annovar/annotate_variation.pl -buildver hg38 -downdb -webfrom annovar refGene ~/annovar/humandb/

  4. The remainder of this guide assumes that ANNOVAR is installed in the directory ~/annovar/ and the annotation databases are downloaded to ~/annovar/humandb/

Usage

ExpansionHunter Denovo case-control or outlier locus-based analysis is performed as described elsewhere in documentation.

The following command annotates an EHdn results file called locus_output.tsv to create an annotated output file locus_output_annotated.tsv.

bash annotate_ehdn.sh \
    --ehdn-results locus_output.tsv \
    --ehdn-annotated-results locus_output_annotated.tsv \
    --annovar-annotate-variation ~/annovar/annotate_variation.pl \
    --annovar-humandb ~/annovar/humandb \
    --annovar-buildver hg19

Here is a description of the required arguments.

Parameter Description
ehdn-results Input filename of EHdn locus analysis results
ehdn-annotated-results Output filename for annotated EHdn locus analysis results
annovar-annotate-variation location of annotate_variation.pl ANNOVAR script
annovar-humandb location of directory containing ANNOVAR refGene annotation database
annovar-builder reference genome build used by ANNOVAR ('hg18', 'hg19' or 'hg38')