https://github.com/asereewit/vadr_hsv

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 command source ~/.zshrc in the terminal.

Download the latest HSV1 (NC001806) and HSV2 (NC001798) VADR models from this github repository.
Run the following v-annotate.pl command (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>

HSV1 and HSV2 VADR models

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

Additional configuration on HSV VADR models

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 NC001806 and 70% HSV2 RefSeq NC001798) 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.pl script 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 ```

Example VADR output

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: ../vadroutput09012023/MG999860vadroutput_09012023

force directory overwrite: yes [-f]

leaving intermediate files on disk: yes [--keep]

specify that alert codes in do not cause FAILure: dupregin,discontn,indfstrp,lowsimis,lowsimil,indf5pst,indf3pst [--alt_pass]

https://github.com/asereewit/vadr_hsv

Science Score: 23.0%

Basic Info

Statistics

Metadata Files

Human alphaherpesvirus 1 (HSV1) and human alphaherpesvirus 2 (HSV2) genome annotation using VADR v1.5.1:

HSV1 and HSV2 VADR models

Additional configuration on HSV VADR models

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: ../vadroutput09012023/MG999860vadroutput_09012023

force directory overwrite: yes [-f]

leaving intermediate files on disk: yes [--keep]

specify that alert codes in do not cause FAILure: dupregin,discontn,indfstrp,lowsimis,lowsimil,indf5pst,indf3pst [--alt_pass]

alert codes in for 'miscnotfailure' features cause miscfeature-ization, not failure: insertnp [--altmnf_yes]

.cm, .minfo, blastn .fa files in $VADRMODELDIR start with key , not 'vadr': NC_001806.vadr [--mkey]

model files are in directory , not in $VADRMODELDIR: /Users/greningerlab/AS/vadrhsv/NC001806 [--mdir]

nmiscftr/TOOMANYMISCFEATURES reported if or more miscfeatures: 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

--- --------- ----- -------- ---- ---- ----

--- --------- ----- -------- ---- ---- ----

Explanation of options used in example HSV1 annotation command

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