UTRannotator

A VEP Plugin to annotate high-impact five prime UTR variants either creating new upstream ORFs or disrupting existing upstream ORFs

Update on Aug 2024

To apply the plugin with the newest version of VEP, please use the plugin version provided by Ensembl (as the compatibility is maintained by Ensembl now): https://github.com/Ensembl/VEP_plugins/blob/release/112/UTRAnnotator.pm.

This repository is an archive for the original version.

Currently, it will annotate whether a small variation (1-5bp) including SNVs, indels and MNVs in 5'UTR would have any of the following molecular consequences:

uAUG_gained: creating a new start codon AUG
uAUG_lost: removing an existing start codon AUG
uSTOP_lost: removing the stop codon of an existing upstream ORF
uSTOP gained: creating a new stop codon within an existing upstream ORF
uFrameShift: creating a frameshift mutation in an existing upstream ORF

Highlights:

The annotation output is transcript-specific not restricted to canonical transcript.

The plugin is applicable to annotate 5'UTR in eukaroyotes.

Background

Requirements

Installation

Usage

Translated small ORF files

Annotation output

Caveats

Background

About the role of 5'UTR variants in human genetic disease:

Whiffin, N., Karczewski, K.J., Zhang, X. et al. Characterising the loss-of-function impact of 5’ untranslated region variants in 15,708 individuals. Nat Commun 11, 2523 (2020). https://doi.org/10.1038/s41467-019-10717-9

About UTRannotator:

Annotating high-impact 5'untranslated region variants with the UTRannotator Zhang, X., Wakeling, M.N., Ware, J.S, Whiffin, N. Bioinformatics; doi: https://academic.oup.com/bioinformatics/advance-article/doi/10.1093/bioinformatics/btaa783/5905476

Requirements

VEP (tested on release-99/202001 and release-100/202005)
PERL (tested on version 5.26.2)

Installation

To use the plugin with VEP, you would need to add the plugin module in Perl's library path. To do this, you could either:

(1) download all the files of this repository to the VEP default path $HOME/.vep/Plugins or

(2) download the repository and add its path to environment variable $PERL5LIB.

e.g. Add this line export PERL5LIB=$PERL5LIB:/path/to/UTRannotator to ~/.bash_profile.

Usage

A written document can be found in this tutorial.

Basic Usage

To run the plugin with VEP, you could the following command line:

vep -i test.vcf --tab -plugin UTRannotator -o test.output

If you are using offline version of VEP, it is essential to use reference genome.

vep -i test.vcf --cache --assembly GRCh38 --fasta /path/to/GRCh38.fa --offline --plugin UTRannotator -o test.output

Note, it's necessary to add option --minimal to transform the alleles into minimal representations if it hasn't been transformed beforehand, especially for variants represented with rs IDs from dbSNP.

Output format

Currently, the output format supports default VEP output format, tab-delimited output and VCF output.

If a variant disrupts multiple uORFs, we will output the annotation for each uORF. The output for each uORF will be concatenated with a logical and symbol &;

Optional Usage

The plugin could also check whether an input variant disrupts a verified translated uORF.

To use this option, users would pass an evidence file of a list of verified translated uORFs as input.

Translated small ORF files

Human (GRCh37/GRCh38)

For translated small ORFs in human, we have curated a list of uORFs previously identified with ribosome profiling from the online repository of small ORFs (www.sorfs.org)

This list is available in the repository:

Genome build GRCh37: uORF_5UTR_GRCh37_PUBLIC.txt

Genome build GRCh38: uORF_5UTR_GRCh38_PUBLIC.txt

The command to use the file is

vep -i test.vcf --tab -plugin UTRannotator,/path/to/uORF_5UTR_GRCh37_PUBLIC.txt -o test.output

To use a customized list of translated uORF, users would curate a tab-delimited txt file with the following columns:

For example:

CHR START_POS GENE STRAND TYPE STOP_POS

19 45971469 FOSB forward five_prime_utr 45971714

START_POS and STOP_POS are the start genomic position and end genomics position of a small ORF respectively.

The following list is a collection of curated translated small ORF files for other species:

Mouse(mm10)

https://github.com/AhmedArslan/orf_mm10 curated by Ahmed Arslan from www.sorfs.org.

Annotation Output

The output annotation from the plugin includes 5 fields:

For any 5'UTR variants, the plugin will first output the number of existing subtype uORFs in the 5'UTR:

Field 1 - existing_InFrame_oORFs : The number of existing inframe overlapping ORFs (inFrame_oORF) already within the 5 prime UTR

Field 2 - existing_OutOfFrame_oORFs : The number of existing out-of-frame overlapping ORFs (OutOfFrame_oORF) already within the 5 prime UTR

Field 3 - existing_uORFs : The number of existing uORFs with a stop codon within the 5 prime UTR

If this 5'UTR is uORF-perturbing, the plugin will output the consequence and detailed annotation of each consequence. Otherwise it will output - :

Field 4 - five_prime_UTR_variant_annotation : Output the annotation of a given 5 prime UTR variant.

Field 5 - five_prime_UTR_variant_consequence : Output the variant consequences of a given 5 prime UTR variant: uAUG_gained, uAUG_lost, uSTOP_gained, uSTOP_lost, uFrameshift.

If a 5'UTR variant perturbs multiple uORFs, the annotation of each uORF will be concatenated with a logical and symbol & for fields five_prime_UTR_variant_consequence and five_prime_UTR_variant_annotation.

Example output (default VEP output)

#Uploaded_variation Location Allele Gene Feature Feature_type Consequence cDNA_position CDS_position Protein_position Amino_acids Codons Existing_variation Extra

5_36877039_CC/A 5:36877039-36877040 A 25836 NM_015384.5 Transcript 5_prime_UTR_variant 169-170 - - - - - IMPACT=MODIFIER;STRAND=1;REFSEQ_MATCH=rseq_mrna_match;existing_InFrame_oORFs=0;existing_OutOfFrame_oORFs=0;existing_uORFs=5;five_prime_UTR_variant_annotation=uFrameShift_Evidence:False,uFrameShift_KozakContext:GCGATGC,uFrameShift_KozakStrength:Moderate,uFrameShift_alt_type:uORF,uFrameShift_alt_type_length:189,uFrameShift_ref_StartDistanceToCDS:324,uFrameShift_ref_type:uORF,uFrameShift_ref_type_length:15;five_prime_UTR_variant_consequence=uFrameShift

The detailed annotation for each consequence

uAUG gained

| Annotations | Data type | Description | |--------------------------|-----------|-------------------------------------------------------------------------------------------------------------------------------------------------| | uAUG_gained_type | String | The type of of 5’ UTR ORF created, described by one of the following: uORF(with a stop codon in 5’UTR), inframe_oORF (inframe and overlapping with CDS),OutOfFrame_oORF (out of frame and overlapping with CDS) | | uAUG_gained_KozakContext | String | The Kozak context sequence of the gained uAUG | | uAUG_gained_KozakStrength | String | The Kozak strength of the gained uAUG, described by one of the following values: Weak, Moderate or Strong. | | uAUG_gained_DistanceToCDS | Integer | The distance (number of nucleotides) between the gained uAUG to CDS | | uAUG_gained_CapDistanceToStart | Integer | The distance (number of nucleotides) between the gained uAUG to the start of 5’UTR | | uAUG_gained_DistanceToSTOP | Integer | The distance (number of nucleotides) between the gained uAUG to STOP codon (scanning through both the 5’UTR and its downstream CDS). If there is no STOP codon found, it would output NA. |

UTRannotator

Install / Use

README

UTRannotator

Update on Aug 2024

Background

Background

Requirements

Installation

Usage

Basic Usage

Output format

Optional Usage

Translated small ORF files

Human (GRCh37/GRCh38)

Mouse(mm10)

Annotation Output

Example output (default VEP output)

The detailed annotation for each consequence

uAUG gained

uAUG lost