A VEP Plugin to annotate high-impact five prime UTR variants either creating new upstream ORFs or disrupting existing upstream ORFs
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.
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
- VEP (tested on release-99/202001 and release-100/202005)
- PERL (tested on version 5.26.2)
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.
A written document can be found in this tutorial.
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.
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 &;
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.
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_starts_ends_GRCh37_PUBLIC.txt
Genome build GRCh38: uORF_starts_ends_GRCh38_PUBLIC.txt
The command to use the file is
vep -i test.vcf --tab -plugin UTRannotator,/path/to/uORF_starts_ends_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:
https://github.com/AhmedArslan/orf_mm10 curated by Ahmed Arslan from www.sorfs.org.
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.
#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
| 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. |
| Annotations | Data type | Description |
|---|---|---|
| uAUG_lost_type | String | The type of 5’ UTR ORF lost, described by one of the following: uORF, inframe_oORF or OutOfFrame_oORF |
| uAUG_lost_KozakContext | String | The Kozak context sequence of the lost uAUG |
| uAUG_lost_KozakStrength | String | The Kozak strength of the lost uAUG, described by one of the following values: Weak, Moderate or Strong. |
| uAUG_lost_CapDistanceToStart | String | The distance between the lost uAUG to the start of the 5'UTR |
| uAUG_lost_DistanceToCDS | Integer | The distance (number of nucleotides) between the lost uAUG to CDS |
| uAUG_lost_DistanceToSTOP | Integer | The distance (number of nucleotides) between the lost uAUG to the nearest stop codon (scanning through both the 5’UTR and its downstream CDS). Output NA if there is no stop codon. |
| uAUG_lost_evidence | Boolean | Whether the uORF disrupted by the lost uAUG has any translation evidence. Output NA if no evidence file provided |
| Annotations | Data type | Description |
|---|---|---|
| uSTOP_lost_AltStop | String | Whether there is an alternative stop codon downstream within 5’ UTR |
| uSTOP_lost_AltStopDistanceToCDS | Integer | The distance between the alternative stop codon (if exists) and CDS. Output NA if there is no alternative stop |
| uSTOP_lost_KozakContext | String | The Kozak context sequence of the disrupted uORF |
| uSTOP_lost_KozakStrength | String | The Kozak strength of the disrupted uORF, described by one of the following values: Weak, Moderate or Strong. |
| uSTOP_lost_FrameWithCDS | String | The frame of the uORF with respect to CDS, described by inFrame or outOfFrame. |
| uSTOP_lost_evidence | Boolean | Whether the uORF disrupted by the lost stop codon has any translation evidence. Output NA if no evidence file provided. |
| Annotations | Data type | Description |
|---|---|---|
| uSTOP_gained_ref_StartDistanceToCDS | Integer | The distance between the uAUG of the disrupting uORF to CDS |
| uSTOP_gained_ref_type | String | The type of uORF being disrupted - any of the following: uORF, inframe_oORF,OutOfFrame_oORF |
| uSTOP_gained_KozakContext | String | The Kozak context sequence of the disrupted uORF |
| uSTOP_gained_KozakStrength | String | The Kozak strength of the disrupted uORF, described by one of the following values: Weak, Moderate or Strong. |
| uSTOP_gained_newSTOPDistanceToCDS | String | The distance between the gained uSTOP to the start of the CDS |
| uSTOP_gained_evidence | Boolean | Whether the disrupted uORF has any translation evidence. Output NA if no evidence file provided. |
| Annotations | Data type | Description |
|---|---|---|
| uFrameshift_ref_type | String | The type of uORF with the reference allele, described by one of following: uORF, inframe_oORF or OutOfFrame_oORF |
| uFrameshift_ref_type_length | Integer | The length of uORF with the reference allele. Output NA if there is no stop codon. |
| uFrameshift_StartDistanceToCDS | Integer | The distance between the start codon of the disrupting uORF and CDS |
| uFrameshift_alt_type | String | The type of uORF with the alternative allele, described by one of following: uORF, inframe_oORF or OutOfFrame_oORF |
| uFrameshift_alt_type_length | Integer | The length of uORF with the alt allele. Output NA if there is no stop codon. |
| uFrameshift_KozakContext | String | The Kozak context sequence of the disrupted uORF |
| uFrameshift_KozakStrength | String | The Kozak strength of the disrupted uORF, described by one of the following values: Weak, Moderate or Strong. |
| uFrameshift_evidence | Boolean | Whether the disrupted uORF has any translation evidence. Output NA if no evidence file provided |
- only tested on SNVs, small indels (1-5bp) and MNV (1-5bp)
- only consider canonical start codon