Human alphaherpesvirus 1 (HSV1) and human alphaherpesvirus 2 (HSV2) genome annotation using VADR v1.5.1
Human alphaherpesvirus 1 (HSV1) and human alphaherpesvirus 2 (HSV2) genome annotation using VADR v1.5.1:
Steps for using VADR for HSV1 and HSV2 annotation:
-
Download and install the latest version of VADR (v1.5.1), following the instructions on this page.
VADR runs on a single processor by default and it can generate a lot of intermediate files. The default maximum number of files a single processor can open on Mac is 256 and sometimes VADR exceeds that. To solve this issue, open the ~/.zshrc file in a text editor and add this line to the .zshrc file
ulimit -n 1024. Then run this commandsource ~/.zshrcin the terminal. -
Download the latest HSV1 (NC_001806) and HSV2 (NC_001798) VADR models from this github repository.
-
Run the following
v-annotate.plcommand (using HSV1 as an example):
v-annotate.pl --mdir <path-to-HSV1-VADR-model-directory> --mkey NC_001806.vadr -s --glsearch -r --alt_pass dupregin,discontn,indfstrn,indfstrp,lowsimis,lowsimil,indf5pst,indf3pst,lowsim5s,lowsim3s --r_lowsimok --alt_mnf_yes insertnp --nmiscftrthr 10 -f --keep <fasta-file-to-annotate> <output-directory-to-create>
The VADR model library for HSV1 was built based on NC_001806 and for HSV2 NC_001798.
The HSV1 VADR model was built using the following v-build.pl command:
v-build.pl --forcelong --skipbuild -f --keep NC_001806 NC_001806
The HSV2 VADR model was built using the following v-build.pl command:
v-build.pl --forcelong --skipbuild -f --keep NC_001798 NC_001798
- The genes and CDS in the inverted repeat regions (LAT, RL1, RL2, RS1)
have
misc_not_failure:"1"in their FEATURE lines in the .minfo file. The HSV genomes are high in GC% (68% in HSV1 RefSeq NC_001806 and 70% HSV2 RefSeq NC_001798) and the number of repeat sequences, particularly in LAT, RL1, RL2, and RS1, that make the sequencing and genome assembly of HSV genomes difficult.misc_not_failure:"1"allows most alerts to annotate those genes and CDS as misc_feature instead of causing failure.
FEATURE NC_001806 type:"gene" coords:"7569..1:-" parent_idx_str:"GBNULL" gene:"LAT" misc_not_failure:"1"
FEATURE NC_001806 type:"gene" coords:"513..1540:+" parent_idx_str:"GBNULL" gene:"RL1" misc_not_failure:"1"
FEATURE NC_001806 type:"CDS" coords:"513..1259:+" parent_idx_str:"GBNULL" gene:"RL1" product:"neurovirulence protein ICP34.5" misc_not_failure:"1"
- Certain CDS have alternative forms/stop codons. To allow this in the VADR model:
- Add extra FEATURE lines for the gene and CDS in the .minfo file like the following:
FEATURE NC_001806 type:"gene" coords:"145198..144124:-" parent_idx_str:"GBNULL" gene:"US10" alternative_ftr_set:"virion protein US10(gene)" alternative_ftr_set_subn:"163"
FEATURE NC_001806 type:"gene" coords:"145198..144144:-" parent_idx_str:"GBNULL" gene:"US10" alternative_ftr_set:"virion protein US10(gene)" alternative_ftr_set_subn:"164"
FEATURE NC_001806 type:"CDS" coords:"145100..144162:-" parent_idx_str:"GBNULL" gene:"US10" product:"virion protein US10" alternative_ftr_set:"virion protein US10(cds)"
FEATURE NC_001806 type:"CDS" coords:"145100..144144:-" parent_idx_str:"GBNULL" gene:"US10" product:"virion protein US10" alternative_ftr_set:"virion protein US10(cds)"
The original CDS for virion protein US10 in HSV1 RefSeq NC_001806 is from genomic position 145100 to 144162 and the alternative is from 145100 to 144144. Note that the "gene" FEATURE line for the alternative CDS has the same coordinates as that of its CDS FEATURE line
The "163" in alternative_ftr_set_subn:"163" refers to the line number minus 1, i.e.
that particular FEATURE line is line 164 in the .minfo file.
- Add the alternative amino acid sequence to the .vadr.protein.fa blastx database using
this
build-add-to-blast.db.plscript from the VADR github repository:
perl build-add-to-blast-db.pl <path-to-minfo-file> <path-to-blast-db-dir> NC_001806 MG999843 144762..143806:- 145100..144144:- <name-for-output-directory>
In this case, NC_001806 is the VADR model accession. MG999843 is the sequence that
has the alternative CDS at 144762..143806:- corresponding to the 145100..144144:-
genomic positions in the VADR model accession.
After successfully running the build-add-to-blast.db.pl script, you should see this
in the .vadr.protein.fa file:
...snip...
>NC_001806.2/145100..144162:-
MIKRRGNVEIRVYYESVRTLRSRSHLKPSDRQQSPGHRVFPGSPGFRDHPENLGNPEYRE
LPETPGYRVTPGIHDNPGLPGSPGLPGSPGLPGSPGPHAPPANHVRLAGLYSPGKYAPLA
SPDPFSPQHGAYARARVGIHTAVRVPPTGSPTHTHLRQDPGDEPTSDDSGLYPLDARALA
HLVMLPADHRAFFRTVVEVSRMCAANVRDPPPPATGAMLGRHARLVHTQWLRANQETSPL
WPWRTAAINFITTMAPRVQTHRHMHDLLMACAFWCCLTHASTCSYAGLYSTHCLHLFGAF
...snip...
>MG999843.1:144762..143806:-/145100..144144:-
MIKRRGNVEIRVYYESVRTLRSRSHLKPSDHQQSPGHRVFPGSPGFRDHPENLGNPEYRE
LPETPGYRVTPGIHDSPGLPGSPGLHXSPGLPGSHGPHAPPANHVRLAGLYSPGKYAPLA
SPDPFSPQDGAYARARVGIHTAVRVPPTGSPTHTHLRHDPGDEPTSDDSGLYPLDARALA
HLVMLPADHRAFFRTVVDVSRMCAANVRDPPPPATGAMLGRHARLVHTQWLRANQETSPL
WPWRTAAINFITTMAPRVQTHRHMHDLLMACAFWCCLTHASTCSYAGLYSTHCLHLFGAF
GCGAPALTPPLCQDNLYP
Below is an example of VADR output when annotating the sequence MG999860 (HSV1).
The VADR log output includes the options used in the v-annotate.pl command,
alert codes and their descriptions, and files saved to output directory.
# v-annotate.pl :: classify and annotate sequences using a model library
# VADR 1.5.1 (Feb 2023)
# - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
# date: Fri Sep 1 16:40:00 2023
# $VADRBIOEASELDIR: /Users/greningerlab/Documents/vadr-install-dir/Bio-Easel
# $VADRBLASTDIR: /Users/greningerlab/Documents/vadr-install-dir/ncbi-blast/bin
# $VADREASELDIR: /Users/greningerlab/Documents/vadr-install-dir/infernal/binaries
# $VADRFASTADIR: /Users/greningerlab/Documents/vadr-install-dir/fasta/bin
# $VADRINFERNALDIR: /Users/greningerlab/Documents/vadr-install-dir/infernal/binaries
# $VADRMODELDIR: /Users/greningerlab/Documents/vadr-install-dir/vadr-models-calici
# $VADRSCRIPTSDIR: /Users/greningerlab/Documents/vadr-install-dir/vadr
#
# sequence file: MG999860.fasta
# output directory: ../vadr_output_09012023/MG999860_vadr_output_09012023
# force directory overwrite: yes [-f]
# leaving intermediate files on disk: yes [--keep]
# specify that alert codes in <s> do not cause FAILure: dupregin,discontn,indfstrp,lowsimis,lowsimil,indf5pst,indf3pst [--alt_pass]
# alert codes in <s> for 'misc_not_failure' features cause misc_feature-ization, not failure: insertnp [--alt_mnf_yes]
# .cm, .minfo, blastn .fa files in $VADRMODELDIR start with key <s>, not 'vadr': NC_001806.vadr [--mkey]
# model files are in directory <s>, not in $VADRMODELDIR: /Users/greningerlab/AS/vadr_hsv/NC_001806 [--mdir]
# nmiscftr/TOO_MANY_MISC_FEATURES reported if <n> or more misc_features: 10 [--nmiscftrthr]
# align with glsearch from the FASTA package, not to a cm with cmalign: yes [--glsearch]
# use top-scoring HSP from blastn to seed the alignment: yes [-s]
# replace stretches of Ns with expected nts, where possible: yes [-r]
# - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
# Validating input ... done. [ 0.1 seconds]
# Preprocessing for N replacement: blastn classification (1 seq) ... done. [ 1.9 seconds]
# Preprocessing for N replacement: coverage determination from blastn results (1 seq) ... done. [ 0.0 seconds]
# Replacing Ns based on results of blastn-based pre-processing ... done. [ 0.0 seconds]
# Classifying sequences with blastn (1 seq) ... done. [ 2.0 seconds]
# Determining sequence coverage from blastn results (1 seq) ... done. [ 0.0 seconds]
# Aligning sequences (NC_001806: 2 seqs) ... done. [ 144.7 seconds]
# Joining alignments from glsearch and blastn for model NC_001806 (1 seq) ... done. [ 0.1 seconds]
# Determining annotation ... done. [ 2.1 seconds]
# Validating proteins with blastx (NC_001806: 1 seq) ... done. [ 26.1 seconds]
# Generating feature table output ... done. [ 0.0 seconds]
# Generating tabular output ... done. [ 0.0 seconds]
#
# Summary of classified sequences:
#
# num num num
#idx model group subgroup seqs pass fail
#--- --------- ----- -------- ---- ---- ----
1 NC_001806 - - 1 0 1
#--- --------- ----- -------- ---- ---- ----
- *all* - - 1 0 1
- *none* - - 0 0 0
#--- --------- ----- -------- ---- ---- ----
#
# Summary of reported alerts:
#
# alert causes short per num num long
#idx code failure description type cases seqs description
#--- -------- ------- --------------------------- -------- ----- ---- -----------
1 dupregin no DUPLICATE_REGIONS sequence 10 1 similarity to a model region occurs more than once
2 discontn no DISCONTINUOUS_SIMILARITY sequence 1 1 not all hits are in the same order in the sequence and the homology model
3 lowsimis no LOW_SIMILARITY sequence 2 1 internal region without significant similarity
4 ambgntrp no N_RICH_REGION_NOT_REPLACED sequence 4 1 N-rich region of unexpected length not replaced
5 indf5pst no INDEFINITE_ANNOTATION_START feature 2 1 protein-based alignment does not extend close enough to nucleotide-based alignment 5' endpoint
6 indf3pst no INDEFINITE_ANNOTATION_END feature 2 1 protein-based alignment does not extend close enough to nucleotide-based alignment 3' endpoint
7 indfstrp no INDEFINITE_STRAND feature 1 1 strand mismatch between protein-based and nucleotide-based predictions
8 lowsimic no LOW_FEATURE_SIMILARITY feature 1 1 region overlapping annotated feature that is or matches a CDS lacks significant similarity
9 lowsimil no LOW_FEATURE_SIMILARITY feature 1 1 long region overlapping annotated feature that does not match a CDS lacks significant similarity
10 ambgnt3f no AMBIGUITY_AT_FEATURE_END feature 2 1 final nucleotide of non-CDS feature is an ambiguous nucleotide
#--- -------- ------- --------------------------- -------- ----- ---- -----------
11 mutendex yes* MUTATION_AT_END feature 1 1 expected stop codon could not be identified, first in-frame stop codon exists 3' of predicted stop position
12 unexleng yes* UNEXPECTED_LENGTH feature 3 1 length of complete coding (CDS or mat_peptide) feature is not a multiple of 3
13 cdsstopn yes* CDS_HAS_STOP_CODON feature 2 1 in-frame stop codon exists 5' of stop position predicted by homology to reference
14 fstukcft yes* POSSIBLE_FRAMESHIFT feature 3 1 possible frameshift in CDS (frame not restored before end)
15 deletinp yes* DELETION_OF_NT feature 1 1 too large of a deletion in protein-based alignment
#--- -------- ------- --------------------------- -------- ----- ---- -----------
#
# Output printed to screen saved in: MG999860_vadr_output_09012023.vadr.log
# List of executed commands saved in: MG999860_vadr_output_09012023.vadr.cmd
# List and description of all output files saved in: MG999860_vadr_output_09012023.vadr.filelist
# copy of input fasta file saved in: MG999860_vadr_output_09012023.vadr.in.fa
# copy of input fasta file with descriptions removed for blastn saved in: MG999860_vadr_output_09012023.vadr.blastn.fa
# esl-seqstat -a output for input fasta file saved in: MG999860_vadr_output_09012023.vadr.seqstat
# Stockholm file for >= 1 possible frameshifts for CDS.42.1 for model NC_001806 saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.42.1.frameshift.stk
# Stockholm file for >= 1 possible frameshifts for CDS.79.1 for model NC_001806 saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.79.1.frameshift.stk
# Stockholm file for >= 1 possible frameshifts for CDS.80.1 for model NC_001806 saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.80.1.frameshift.stk
# Stockholm file for >= 1 possible frameshifts for CDS.81.1 for model NC_001806 saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.81.1.frameshift.stk
# model NC_001806 feature gene#1 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.1.fa
# model NC_001806 feature gene#2 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.2.fa
# model NC_001806 feature CDS#1 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.1.fa
# model NC_001806 feature gene#3 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.3.fa
# model NC_001806 feature CDS#2 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.2.fa
# model NC_001806 feature gene#4 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.4.fa
# model NC_001806 feature CDS#3 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.3.fa
# model NC_001806 feature gene#5 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.5.fa
# model NC_001806 feature CDS#4 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.4.fa
# model NC_001806 feature gene#6 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.6.fa
# model NC_001806 feature CDS#5 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.5.fa
# model NC_001806 feature gene#7 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.7.fa
# model NC_001806 feature gene#8 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.8.fa
# model NC_001806 feature CDS#6 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.6.fa
# model NC_001806 feature CDS#7 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.7.fa
# model NC_001806 feature gene#9 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.9.fa
# model NC_001806 feature CDS#8 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.8.fa
# model NC_001806 feature gene#10 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.10.fa
# model NC_001806 feature CDS#9 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.9.fa
# model NC_001806 feature gene#11 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.11.fa
# model NC_001806 feature gene#12 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.12.fa
# model NC_001806 feature CDS#10 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.10.fa
# model NC_001806 feature CDS#11 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.11.fa
# model NC_001806 feature gene#13 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.13.fa
# model NC_001806 feature CDS#12 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.12.fa
# model NC_001806 feature gene#14 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.14.fa
# model NC_001806 feature gene#15 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.15.fa
# model NC_001806 feature gene#16 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.16.fa
# model NC_001806 feature gene#17 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.17.fa
# model NC_001806 feature gene#18 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.18.fa
# model NC_001806 feature CDS#13 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.13.fa
# model NC_001806 feature CDS#14 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.14.fa
# model NC_001806 feature CDS#15 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.15.fa
# model NC_001806 feature CDS#16 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.16.fa
# model NC_001806 feature CDS#17 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.17.fa
# model NC_001806 feature gene#19 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.19.fa
# model NC_001806 feature gene#20 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.20.fa
# model NC_001806 feature CDS#18 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.18.fa
# model NC_001806 feature CDS#19 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.19.fa
# model NC_001806 feature gene#21 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.21.fa
# model NC_001806 feature gene#22 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.22.fa
# model NC_001806 feature gene#23 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.23.fa
# model NC_001806 feature CDS#20 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.20.fa
# model NC_001806 feature CDS#21 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.21.fa
# model NC_001806 feature CDS#22 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.22.fa
# model NC_001806 feature gene#24 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.24.fa
# model NC_001806 feature gene#25 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.25.fa
# model NC_001806 feature gene#26 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.26.fa
# model NC_001806 feature CDS#23 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.23.fa
# model NC_001806 feature CDS#24 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.24.fa
# model NC_001806 feature CDS#25 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.25.fa
# model NC_001806 feature gene#27 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.27.fa
# model NC_001806 feature CDS#26 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.26.fa
# model NC_001806 feature gene#28 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.28.fa
# model NC_001806 feature CDS#27 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.27.fa
# model NC_001806 feature gene#29 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.29.fa
# model NC_001806 feature CDS#28 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.28.fa
# model NC_001806 feature gene#30 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.30.fa
# model NC_001806 feature CDS#29 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.29.fa
# model NC_001806 feature gene#31 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.31.fa
# model NC_001806 feature CDS#30 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.30.fa
# model NC_001806 feature gene#32 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.32.fa
# model NC_001806 feature CDS#31 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.31.fa
# model NC_001806 feature gene#33 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.33.fa
# model NC_001806 feature CDS#32 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.32.fa
# model NC_001806 feature gene#34 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.34.fa
# model NC_001806 feature gene#35 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.35.fa
# model NC_001806 feature CDS#33 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.33.fa
# model NC_001806 feature CDS#34 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.34.fa
# model NC_001806 feature gene#36 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.36.fa
# model NC_001806 feature CDS#35 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.35.fa
# model NC_001806 feature gene#37 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.37.fa
# model NC_001806 feature CDS#36 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.36.fa
# model NC_001806 feature gene#38 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.38.fa
# model NC_001806 feature gene#39 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.39.fa
# model NC_001806 feature CDS#37 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.37.fa
# model NC_001806 feature CDS#38 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.38.fa
# model NC_001806 feature gene#40 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.40.fa
# model NC_001806 feature CDS#39 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.39.fa
# model NC_001806 feature gene#41 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.41.fa
# model NC_001806 feature CDS#40 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.40.fa
# model NC_001806 feature gene#42 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.42.fa
# model NC_001806 feature CDS#41 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.41.fa
# model NC_001806 feature gene#43 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.43.fa
# model NC_001806 feature CDS#42 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.42.fa
# model NC_001806 feature gene#44 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.44.fa
# model NC_001806 feature CDS#43 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.43.fa
# model NC_001806 feature gene#45 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.45.fa
# model NC_001806 feature CDS#44 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.44.fa
# model NC_001806 feature gene#46 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.46.fa
# model NC_001806 feature CDS#45 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.45.fa
# model NC_001806 feature gene#47 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.47.fa
# model NC_001806 feature CDS#46 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.46.fa
# model NC_001806 feature gene#48 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.48.fa
# model NC_001806 feature CDS#47 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.47.fa
# model NC_001806 feature gene#49 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.49.fa
# model NC_001806 feature gene#50 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.50.fa
# model NC_001806 feature CDS#48 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.48.fa
# model NC_001806 feature CDS#49 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.49.fa
# model NC_001806 feature gene#51 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.51.fa
# model NC_001806 feature gene#52 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.52.fa
# model NC_001806 feature CDS#50 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.50.fa
# model NC_001806 feature CDS#51 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.51.fa
# model NC_001806 feature gene#53 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.53.fa
# model NC_001806 feature CDS#52 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.52.fa
# model NC_001806 feature gene#54 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.54.fa
# model NC_001806 feature CDS#53 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.53.fa
# model NC_001806 feature gene#55 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.55.fa
# model NC_001806 feature gene#56 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.56.fa
# model NC_001806 feature CDS#54 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.54.fa
# model NC_001806 feature CDS#55 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.55.fa
# model NC_001806 feature gene#57 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.57.fa
# model NC_001806 feature CDS#56 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.56.fa
# model NC_001806 feature gene#58 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.58.fa
# model NC_001806 feature gene#59 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.59.fa
# model NC_001806 feature CDS#57 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.57.fa
# model NC_001806 feature CDS#58 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.58.fa
# model NC_001806 feature gene#60 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.60.fa
# model NC_001806 feature CDS#59 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.59.fa
# model NC_001806 feature gene#61 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.61.fa
# model NC_001806 feature CDS#60 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.60.fa
# model NC_001806 feature gene#62 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.62.fa
# model NC_001806 feature CDS#61 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.61.fa
# model NC_001806 feature gene#63 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.63.fa
# model NC_001806 feature CDS#62 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.62.fa
# model NC_001806 feature gene#64 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.64.fa
# model NC_001806 feature CDS#63 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.63.fa
# model NC_001806 feature gene#65 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.65.fa
# model NC_001806 feature CDS#64 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.64.fa
# model NC_001806 feature gene#66 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.66.fa
# model NC_001806 feature CDS#65 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.65.fa
# model NC_001806 feature gene#67 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.67.fa
# model NC_001806 feature gene#68 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.68.fa
# model NC_001806 feature CDS#66 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.66.fa
# model NC_001806 feature gene#69 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.69.fa
# model NC_001806 feature CDS#67 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.67.fa
# model NC_001806 feature gene#70 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.70.fa
# model NC_001806 feature CDS#68 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.68.fa
# model NC_001806 feature gene#71 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.71.fa
# model NC_001806 feature CDS#69 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.69.fa
# model NC_001806 feature gene#72 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.72.fa
# model NC_001806 feature CDS#70 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.70.fa
# model NC_001806 feature gene#73 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.73.fa
# model NC_001806 feature CDS#71 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.71.fa
# model NC_001806 feature gene#74 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.74.fa
# model NC_001806 feature CDS#72 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.72.fa
# model NC_001806 feature gene#75 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.75.fa
# model NC_001806 feature CDS#73 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.73.fa
# model NC_001806 feature gene#76 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.76.fa
# model NC_001806 feature CDS#74 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.74.fa
# model NC_001806 feature gene#77 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.77.fa
# model NC_001806 feature CDS#75 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.75.fa
# model NC_001806 feature gene#78 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.78.fa
# model NC_001806 feature CDS#76 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.76.fa
# model NC_001806 feature gene#79 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.79.fa
# model NC_001806 feature CDS#77 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.77.fa
# model NC_001806 feature gene#80 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.80.fa
# model NC_001806 feature CDS#78 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.78.fa
# model NC_001806 feature gene#81 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.81.fa
# model NC_001806 feature gene#82 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.82.fa
# model NC_001806 feature gene#83 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.83.fa
# model NC_001806 feature gene#84 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.84.fa
# model NC_001806 feature CDS#79 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.79.fa
# model NC_001806 feature CDS#80 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.80.fa
# model NC_001806 feature CDS#81 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.81.fa
# model NC_001806 feature CDS#82 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.82.fa
# model NC_001806 feature gene#85 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.gene.85.fa
# model NC_001806 feature CDS#83 predicted seqs saved in: MG999860_vadr_output_09012023.vadr.NC_001806.CDS.83.fa
# model NC_001806 replaced sequence alignment (stockholm) saved in: MG999860_vadr_output_09012023.vadr.NC_001806.align.rpstk
# model NC_001806 full original sequence alignment (stockholm) saved in: MG999860_vadr_output_09012023.vadr.NC_001806.align.stk
# model NC_001806 full replaced sequence alignment (afa) saved in: MG999860_vadr_output_09012023.vadr.NC_001806.align.rpafa
# model NC_001806 full original sequence alignment (afa) saved in: MG999860_vadr_output_09012023.vadr.NC_001806.align.afa
# 5 column feature table output for passing sequences saved in: MG999860_vadr_output_09012023.vadr.pass.tbl
# 5 column feature table output for failing sequences saved in: MG999860_vadr_output_09012023.vadr.fail.tbl
# list of passing sequences saved in: MG999860_vadr_output_09012023.vadr.pass.list
# list of failing sequences saved in: MG999860_vadr_output_09012023.vadr.fail.list
# list of alerts in the feature tables saved in: MG999860_vadr_output_09012023.vadr.alt.list
# fasta file with passing sequences saved in: MG999860_vadr_output_09012023.vadr.pass.fa
# fasta file with failing sequences saved in: MG999860_vadr_output_09012023.vadr.fail.fa
# per-sequence tabular annotation summary file saved in: MG999860_vadr_output_09012023.vadr.sqa
# per-sequence tabular classification summary file saved in: MG999860_vadr_output_09012023.vadr.sqc
# per-feature tabular summary file saved in: MG999860_vadr_output_09012023.vadr.ftr
# per-model-segment tabular summary file saved in: MG999860_vadr_output_09012023.vadr.sgm
# per-model tabular summary file saved in: MG999860_vadr_output_09012023.vadr.mdl
# per-alert tabular summary file saved in: MG999860_vadr_output_09012023.vadr.alt
# alert count tabular summary file saved in: MG999860_vadr_output_09012023.vadr.alc
# alignment doctoring tabular summary file saved in: MG999860_vadr_output_09012023.vadr.dcr
# seed alignment summary file (-s) saved in: MG999860_vadr_output_09012023.vadr.sda
# replaced stretches of Ns summary file (-r) saved in: MG999860_vadr_output_09012023.vadr.rpn
#
# All output files created in directory ./../vadr_output_09012023/MG999860_vadr_output_09012023/
#
# Elapsed time: 00:02:57.07
# hh:mm:ss
#
[ok]
Detailed information on how to interpret VADR alerts are here.
Particularly useful files:
.pass.fa: fasta sequence that passes annotation
.pass.tbl: feature table format of annotation for sequence that passes annotation
.fail.fa: fasta sequence that fails annotation
.fail.tbl: feature table format of annotation for sequence that fails annotation (note that the end of the file has error messages)
.alt.list: list of alert codes that cause annotation failure
.alt: list of all alert codes
.align.stk: stockholm alignment of input sequence to VADR model sequence\
The options used in the example command are recommended, but other options might
be more appropriate depending on genome assembly methods and quality of the
genomes. A complete list of options for v-annotate.pl can be found here.
| option | explanation |
|---|---|
--mdir <path-to-HSV1-VADR-model-directory> |
specify that the model to use are in directory path-to-HSV1-VADR-model-directory |
--mkey NC_001806.vadr |
use the model files with prefix NC_001806.vadr in the directory from --mdir |
-s |
turn on the seed acceleration heuristic: use the max length ungapped region from blastn to seed the alignment, required for HSV annotation |
--glsearch |
use the glsearch program from the FASTA package for the alignment stage, required for HSV annotation |
-r |
turn on the replace-N strategy: replace stretches of Ns with expected nucleotides, where possible |
--alt_pass dupregin,discontn,indfstrn,indfstrp,lowsimis,lowsimil,indf5pst,indf3pst |
specify that these alerts, mostly due to inverted repeats and Ns in the sequence, do NOT cause annotation failure |
--r_lowsimok |
with -r, do not report lowsim{5,3,i}s within identified N-rich regions |
--alt_mnf_yes insertnp |
specify that features with misc_not_failure:"1" in .minfo file (LAT, RL1, RL2, RS1) also allow insertnp alert; misc_not_failure:"1" allows insertnn alert but not insertnp alert, which are raised together usually |
--nmiscftrthr 10 |
increase the number of misc_feature annotation allowed in a sequence to 10 due to the use of misc_not_failure:"1" in LAT, RL1, RL2, RS2 |
-f |
overwrite output directory if it exists |
--keep |
keep all intermediate files, useful for debugging |
<fasta-file-to-annotate> |
input fasta sequence to be annotate |
<output-directory-to-create> |
output directory |
- VADR README
- VADR installation instructions
v-build.plexample usage and command-line optionsv-annotate.plexample usage, command-line options and alert information- Explanations and examples of
v-annotate.pldetailed alert and error messages- Output fields with detailed alert and error messages
- Explanation of sequence and model coordinate fields in
.altfiles toy50toy model used in examples of alert messages- Examples of different alert types and corresponding
.altoutput - Posterior probability annotation in VADR output Stockholm alignments
- VADR output file formats
- The recommended citation for using VADR is: Alejandro A Schäffer, Eneida L Hatcher, Linda Yankie, Lara Shonkwiler, J Rodney Brister, Ilene Karsch-Mizrachi, Eric P Nawrocki; VADR: validation and annotation of virus sequence submissions to GenBank. BMC Bioinformatics 21, 211 (2020). https://doi.org/10.1186/s12859-020-3537-3
- https://github.com/ncbi/vadr/wiki/Mpox-virus-annotation