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Variant Annotation

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Annotation

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ANNOVAR

ANNOVAR is a simple and efficient command line tool for variant annotation.

In this tutorial, we will use ANNOVAR to annotate the variants in our summary statistics (hg19).

Install

Download ANNOVAR from here (registration required; freely available to personal, academic and non-profit use only.)

You will receive an email with the download link after registration. Download it and decompress:

tar -xvzf annovar.latest.tar.gz

For refGene annotation for hg19, we do not need to download additional files.

Format input file

The default input file for ANNOVAR is a 1-based coordinate file.

We will only use the first 100000 variants as an example.

annovar_input

awk 'NR>1 && NR<100000 {print $1,$2,$2,$4,$5}' ../06_Association_tests/1kgeas.B1.glm.firth > annovar_input.txt

head annovar_input.txt 
1 13273 13273 G C
1 14599 14599 T A
1 14604 14604 A G
1 14930 14930 A G
1 69897 69897 T C
1 86331 86331 A G
1 91581 91581 G A
1 122872 122872 T G
1 135163 135163 C T
1 233473 233473 C G

With -vcfinput option, ANNOVAR can accept input files in VCF format.

Annotation

Annotate the variants with gene information.

A minimal example of annotation using refGene

input=annovar_input.txt
humandb=/home/he/tools/annovar/annovar/humandb
table_annovar.pl ${input} ${humandb} -buildver hg19 -out myannotation -remove -protocol refGene     -operation g -nastring . -polish

Chr Start   End Ref Alt Func.refGene    Gene.refGene    GeneDetail.refGene  ExonicFunc.refGene  AAChange.    refGene
1   13273   13273   G   C   ncRNA_exonic    DDX11L1;LOC102725121    .   .   .
1   14599   14599   T   A   ncRNA_exonic    WASH7P  .   .   .
1   14604   14604   A   G   ncRNA_exonic    WASH7P  .   .   .
1   14930   14930   A   G   ncRNA_intronic  WASH7P  .   .   .
1   69897   69897   T   C   exonic  OR4F5   .   synonymous SNV  OR4F5:NM_001005484:exon1:c.T807C:p.S269S
1   86331   86331   A   G   intergenic  OR4F5;LOC729737 dist=16323;dist=48442   .   .
1   91581   91581   G   A   intergenic  OR4F5;LOC729737 dist=21573;dist=43192   .   .
1   122872  122872  T   G   intergenic  OR4F5;LOC729737 dist=52864;dist=11901   .   .
1   135163  135163  C   T   ncRNA_exonic    LOC729737   .   .   .

Additional databases

ANNOVAR supports a wide range of commonly used databases including dbsnp , dbnsfp, clinvar, gnomad, 1000g, cadd and so forth. For details, please check ANNOVAR's official documents

You can check the Table Name listed in the link above and download the database you need using the following command.

Example: Downloading avsnp150 for hg19 from ANNOVAR

annotate_variation.pl -buildver hg19 -downdb -webfrom annovar avsnp150 humandb/

An example of annotation using multiple databases

# input file is in vcf format
table_annovar.pl \
  ${in_vcf} \
  ${humandb} \
  -buildver hg19 \
  -protocol refGene,avsnp150,clinvar_20200316,gnomad211_exome \
  -operation g,f,f,f \
  -remove \
  -out ${out_prefix} \ 
  -vcfinput

VEP (under construction)

Install

git clone https://github.com/Ensembl/ensembl-vep.git
cd ensembl-vep
perl INSTALL.pl

Hello! This installer is configured to install v108 of the Ensembl API for use by the VEP.
It will not affect any existing installations of the Ensembl API that you may have.

It will also download and install cache files from Ensembl's FTP server.

Checking for installed versions of the Ensembl API...done

Setting up directories
Destination directory ./Bio already exists.
Do you want to overwrite it (if updating VEP this is probably OK) (y/n)? y
 - fetching BioPerl
 - unpacking ./Bio/tmp/release-1-6-924.zip
 - moving files

Downloading required Ensembl API files
 - fetching ensembl
 - unpacking ./Bio/tmp/ensembl.zip
 - moving files
 - getting version information
 - fetching ensembl-variation
 - unpacking ./Bio/tmp/ensembl-variation.zip
 - moving files
 - getting version information
 - fetching ensembl-funcgen
 - unpacking ./Bio/tmp/ensembl-funcgen.zip
 - moving files
 - getting version information
 - fetching ensembl-io
 - unpacking ./Bio/tmp/ensembl-io.zip
 - moving files
 - getting version information

Testing VEP installation
 - OK!

The VEP can either connect to remote or local databases, or use local cache files.
Using local cache files is the fastest and most efficient way to run the VEP
Cache files will be stored in /home/he/.vep
Do you want to install any cache files (y/n)? y

The following species/files are available; which do you want (specify multiple separated by spaces or 0 for all): 
1 : acanthochromis_polyacanthus_vep_108_ASM210954v1.tar.gz (69 MB)
2 : accipiter_nisus_vep_108_Accipiter_nisus_ver1.0.tar.gz (55 MB)
...
466 : homo_sapiens_merged_vep_108_GRCh37.tar.gz (16 GB)
467 : homo_sapiens_merged_vep_108_GRCh38.tar.gz (26 GB)
468 : homo_sapiens_refseq_vep_108_GRCh37.tar.gz (13 GB)
469 : homo_sapiens_refseq_vep_108_GRCh38.tar.gz (22 GB)
470 : homo_sapiens_vep_108_GRCh37.tar.gz (14 GB)
471 : homo_sapiens_vep_108_GRCh38.tar.gz (22 GB)

  Total: 221 GB for all 471 files

? 470
 - downloading https://ftp.ensembl.org/pub/release-108/variation/indexed_vep_cache/homo_sapiens_vep_108_GRCh37.tar.gz