Biowulf High Performance Computing at the NIH
VADR: Viral Annotation DefineR.

VADR is a suite of tools for classifying and analyzing sequences homologous to a set of reference models of viral genomes or gene families. It has been mainly tested for analysis of Norovirus, Dengue, and SARS-CoV-2 virus sequences in preparation for submission to the GenBank database.

References:

Documentation
Important Notes

Interactive job
Interactive jobs should be used for debugging, graphics, or applications that cannot be run as batch jobs.

Allocate an interactive session and run the program. Sample session:

[user@biowulf]$ sinteractive 
[user@cn4471 ~]$ module load vadr 
[+] Loading hmmer  3.3.2  on cn0847
[+] Loading perl 5.24.3 on cn0847
[+] Loading vadr / 1.1.3  ...
Copy sample data to your current folder:
[user@cn4471 ~]$ cp $VADR_DATA/* .
Preprocess the data with samtools:
[user@cn4471 ~]$ v-build.pl -h
...
# v-build.pl :: build homology model of a single sequence for feature annotation
# VADR 1.1.3 (Feb 2021)
# - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
# date:    Mon Mar 29 12:40:22 2021
#
Usage: v-build.pl [-options] <accession> <path to output directory to create>

basic options:
  -f             : force; if dir <output directory> exists, overwrite it
  -v             : be verbose; output commands to stdout as they're run
  --stk <s>      : read single sequence stockholm 'alignment' from <s>
  --infa <s>     : read single sequence fasta file from <s>, don't fetch it
  --inft <s>     : read feature table file from <s>, don't fetch it
  --ftfetch1     : fetch feature table with efetch -format ft
  --ftfetch2     : fetch feature table with efetch -format gbc | xml2tbl
  --gb           : parse a genbank file, not a feature table file
  --ingb <s>     : read genbank file from <s>, don't fetch it
  --addminfo <s> : add feature info from model info file <s>
  --forcelong    : allow long models > 25Kb in length
  --keep         : do not remove intermediate files, keep them all on disk

options for controlling what feature types are stored in model info file
[default set is: CDS,gene,mat_peptide]:
  --fall      : store info for all feature types (except those in --fskip)
  --fadd <s>  : also store feature types in comma separated string <s>
  --fskip <s> : do not store info for feature types in comma separated string <s>

options for controlling what qualifiers are stored in model info file
[default set is:product,gene,exception]:
  --qall        : store info for all qualifiers (except those in --qskip)
  --qadd <s>    : also store info for qualifiers in comma separated string <s>
  --qftradd <s> : --qadd <s2> only applies for feature types in comma separated string <s>
  --qskip <s>   : do not store info for qualifiers in comma separated string <s>
  --noaddgene   : do not add gene qualifiers from gene features to overlapping features

options for including additional model attributes:
  --group <s>    : specify model group is <s>
  --subgroup <s> : specify model subgroup is <s>

options for controlling CDS translation step:
  --ttbl <n> : use NCBI translation table <n> to translate CDS [1]

options for controlling cmbuild step:
  --cmn <n>       : set number of seqs for glocal fwd HMM calibration to <n>
  --cmp7ml        : set CM's filter p7 HMM as the ML p7 HMM
  --cmere <x>     : set CM relative entropy target to <x>
  --cmeset <x>    : set CM eff seq # for CM to <x>
  --cmemaxseq <x> : set CM maximum alowed eff seq # for CM to <x>
  --cminfile <s>  : read cmbuild options from file <s>

options for skipping stages:
  --skipbuild : skip the cmbuild and blastn db creation steps
  --onlyurl   : output genbank file url for accession and exit

optional output files:
  --ftrinfo : create file with internal feature information
  --sgminfo : create file with internal segment information

other expert options:
  --execname <s> : define executable name of this script as <s>
[user@cn4471 ~]$ v-build.pl NC_039897 NC_039897
# v-build.pl :: build homology model of a single sequence for feature annotation
# VADR 1.1.3 (Feb 2021)
# - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
# date:              Tue Mar 30 14:51:04 2021
# $VADRBLASTDIR:     /usr/local/apps/VADR/1.1.3/vadr-vadr-1.1.3/ncbi-blast/bin
# $VADREASELDIR:     /usr/local/apps/VADR/1.1.3/vadr-vadr-1.1.3/Bio-Easel/src/easel/miniapps
# $VADRINFERNALDIR:  /usr/local/apps/VADR/1.1.3/vadr-vadr-1.1.3/infernal/binaries
# $VADRSCRIPTSDIR:   /usr/local/apps/VADR/1.1.3/vadr-vadr-1.1.3/vadr
#
# accession/model name:  NC_039897
# output directory:      NC_039897
# - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
# Fetching FASTA file                                          ... done. [    3.3 seconds]
# Parsing FASTA file                                           ... done. [    0.0 seconds]
# Fetching feature table file                                  ... done. [    3.9 seconds]
# Parsing feature table file                                   ... done. [    0.0 seconds]
# Fetching and parsing protein feature table file(s)           ... done. [   11.8 seconds]
# Pruning data read from GenBank                               ... done. [    0.0 seconds]
# Reformatting FASTA file to Stockholm file                    ... done. [    0.0 seconds]
# Finalizing feature information                               ... done. [    0.0 seconds]
# Translating CDS                                              ... done. [    0.0 seconds]
# Building BLAST protein database                              ... done. [    0.2 seconds]
# Building HMMER protein database                              ... done. [    2.6 seconds]
# Building CM (should take roughly 10-30 minutes)              ... done. [  749.1 seconds]
# Pressing CM file                                             ... done. [    0.2 seconds]
# Building BLAST nucleotide database of CM consensus           ... done. [    0.4 seconds]
# Creating model info file                                     ... done. [    0.0 seconds]
#
# Output printed to screen saved in:                                           NC_039897.vadr.log
# List of executed commands saved in:                                          NC_039897.vadr.cmd
# List and description of all output files saved in:                           NC_039897.vadr.filelist
# fasta file for NC_039897 saved in:                                           NC_039897.vadr.fa
# feature table format file for NC_039897 saved in:                            NC_039897.vadr.tbl
# feature table format file for YP_009538340.1 saved in:                       NC_039897.vadr.YP_009538340.1.tbl
# feature table format file for YP_009538341.1 saved in:                       NC_039897.vadr.YP_009538341.1.tbl
# feature table format file for YP_009538342.1 saved in:                       NC_039897.vadr.YP_009538342.1.tbl
# Stockholm alignment file for NC_039897 saved in:                             NC_039897.vadr.stk
# fasta sequence file for CDS from NC_039897 saved in:                         NC_039897.vadr.cds.fa
# fasta sequence file for translated CDS from NC_039897 saved in:              NC_039897.vadr.protein.fa
# BLAST db .phr file for NC_039897 saved in:                                   NC_039897.vadr.protein.fa.phr
# BLAST db .pin file for NC_039897 saved in:                                   NC_039897.vadr.protein.fa.pin
# BLAST db .psq file for NC_039897 saved in:                                   NC_039897.vadr.protein.fa.psq
# BLAST db .pdb file for NC_039897 saved in:                                   NC_039897.vadr.protein.fa.pdb
# BLAST db .pot file for NC_039897 saved in:                                   NC_039897.vadr.protein.fa.pot
# BLAST db .ptf file for NC_039897 saved in:                                   NC_039897.vadr.protein.fa.ptf
# BLAST db .pto file for NC_039897 saved in:                                   NC_039897.vadr.protein.fa.pto
# HMMER model db file for NC_039897 saved in:                                  NC_039897.vadr.protein.hmm
# hmmbuild build output (concatenated) saved in:                               NC_039897.vadr.protein.hmmbuild
# binary HMM and p7 HMM filter file saved in:                                  NC_039897.vadr.protein.hmm.h3m
# SSI index for binary HMM file saved in:                                      NC_039897.vadr.protein.hmm.h3i
# optimized p7 HMM filters (MSV part) saved in:                                NC_039897.vadr.protein.hmm.h3f
# optimized p7 HMM filters (remainder) saved in:                               NC_039897.vadr.protein.hmm.h3p
# hmmpress output file saved in:                                               NC_039897.vadr.hmmpress
# CM file saved in:                                                            NC_039897.vadr.cm
# cmbuild output file saved in:                                                NC_039897.vadr.cmbuild
# binary CM and p7 HMM filter file saved in:                                   NC_039897.vadr.cm.i1m
# SSI index for binary CM file saved in:                                       NC_039897.vadr.cm.i1i
# optimized p7 HMM filters (MSV part) saved in:                                NC_039897.vadr.cm.i1f
# optimized p7 HMM filters (remainder) saved in:                               NC_039897.vadr.cm.i1p
# cmpress output file saved in:                                                NC_039897.vadr.cmpress
# fasta sequence file with cmemit consensus sequence for NC_039897 saved in:   NC_039897.vadr.nt.fa
# BLAST db .nhr file for NC_039897 saved in:                                   NC_039897.vadr.nt.fa.nhr
# BLAST db .nin file for NC_039897 saved in:                                   NC_039897.vadr.nt.fa.nin
# BLAST db .nsq file for NC_039897 saved in:                                   NC_039897.vadr.nt.fa.nsq
# BLAST db .ndb file for NC_039897 saved in:                                   NC_039897.vadr.nt.fa.ndb
# BLAST db .not file for NC_039897 saved in:                                   NC_039897.vadr.nt.fa.not
# BLAST db .ntf file for NC_039897 saved in:                                   NC_039897.vadr.nt.fa.ntf
# BLAST db .nto file for NC_039897 saved in:                                   NC_039897.vadr.nt.fa.nto
# VADR 'model info' format file for NC_039897 saved in:                        NC_039897.vadr.minfo
#
# All output files created in directory ./NC_039897/
#
# Elapsed time:  00:12:51.55
#                hh:mm:ss
#
[ok]
[user@cn4471 ~]$ v-annotate.pl -h 
# v-annotate.pl :: classify and annotate sequences using a CM library
# VADR 1.1.3 (Feb 2021)
# - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
# date:    Tue Mar 30 15:17:51 2021
#
Usage: v-annotate.pl [-options] <fasta file to annotate> <output directory to create>

basic options:
  -f             : force; if output dir exists, overwrite it
  -v             : be verbose; output commands to stdout as they're run
  --atgonly      : only consider ATG a valid start codon
  --minpvlen <n> : min CDS/mat_peptide/gene length for feature table output and protein validation is <n> [30]
  --keep         : do not remove intermediate files, keep them all on disk

options for specifying classification:
  --group <s>    : set expected classification of all seqs to group <s>
  --subgroup <s> : set expected classification of all seqs to subgroup <s>

options for controlling severity of alerts:
  --alt_list        : output summary of all alerts and exit
  --alt_pass <s>    : specify that alert codes in comma-separated <s> do not cause FAILure
  --alt_fail <s>    : specify that alert codes in comma-separated <s> do cause FAILure
  --alt_mnf_yes <s> : alert codes in <s> for 'misc_not_failure' features cause misc_feature-ization, not failure
  --alt_mnf_no <s>  : alert codes in <s> for 'misc_not_failure' features cause failure, not misc-feature-ization
  --ignore_mnf      : ignore non-zero 'misc_not_feature' values in .minfo file, set to 0 for all features/models

options related to model files:
  -m <s>      : use CM file <s> instead of default
  -a <s>      : use HMM file <s> instead of default
  -i <s>      : use model info file <s> instead of default
  -n <s>      : use blastn db file <s> instead of default
  -x <s>      : blastx dbs are in dir <s>, instead of default
  --mkey <s>  : .cm, .minfo, blastn .fa files in $VADRMODELDIR start with key <s>, not 'vadr'
  --mdir <s>  : model files are in directory <s>, not in $VADRMODELDIR
  --mlist <s> : only use models listed in file <s>

options for controlling output feature table:
  --nomisc        : in feature table for failed seqs, never change feature type to misc_feature
  --notrim        : in feature table, don't trim coords due to Ns (for any feature types)
  --noftrtrim <s> : in feature table, don't trim coords due to Ns for feature types in comma-delmited <s>
  --noprotid      : in feature table, don't add protein_id for CDS and mat_peptides
  --forceprotid   : in feature table, force protein_id value to be sequence name, then idx

options for controlling thresholds related to alerts:
  --lowsc <x>       : lowscore/LOW_SCORE bits per nucleotide threshold is <x> [0.3]
  --indefclass <x>  : indfcls/INDEFINITE_CLASSIFICATION bits per nucleotide diff threshold is <x> [0.03]
  --incspec <x>     : inc{group,subgrp}/INCORRECT_{GROUP,SUBGROUP} bits/nt threshold is <x> [0.2]
  --lowcov <x>      : lowcovrg/LOW_COVERAGE fractional coverage threshold is <x> [0.9]
  --dupregolp <n>   : dupregin/DUPLICATE_REGIONS minimum model overlap is <n> [20]
  --dupregsc <x>    : dupregin/DUPLICATE_REGIONS minimum bit score is <x> [10]
  --indefstr <x>    : indfstrn/INDEFINITE_STRAND minimum weaker strand bit score is <x> [25]
  --lowsim5term <n> : lowsim5{s,f}/LOW_{FEATURE_}SIMILARITY_START minimum length is <n> [15]
  --lowsim3term <n> : lowsim3{s,f}/LOW_{FEATURE_}SIMILARITY_END minimum length is <n> [15]
  --lowsimint <n>   : lowsimi{s,f}/LOW_{FEATURE_}SIMILARITY (internal) minimum length is <n> [1]
  --biasfract <x>   : biasdseq/BIASED_SEQUENCE fractional threshold is <x> [0.25]
  --indefann <x>    : indf{5,3}loc/'INDEFINITE_ANNOTATION_{START,END} non-mat_peptide min allowed post probability is <x> [0.8]
  --indefann_mp <x> : indf{5,3}loc/'INDEFINITE_ANNOTATION_{START,END} mat_peptide min allowed post probability is <x> [0.6]
  --fstminnt <n>    : fst{hi,lo}cnf/POSSIBLE_FRAMESHIFT_{HIGH,LOW}_CONF max allowed frame disagreement nt length w/o alert is <n> [6]
  --fsthighthr <x>  : fsthicnf/POSSIBLE_FRAMESHIFT_HIGH_CONF minimum average probability for alert is <x> [0.8]
  --fstlowthr <x>   : fstlocnf/POSSIBLE_FRAMESHIFT_LOW_CONF minimum average probability for alert is <x> [0.3]
  --xalntol <n>     : indf{5,3}{st,lg}/INDEFINITE_ANNOTATION_{START,END} max allowed nt diff blastx start/end is <n> [5]
  --xmaxins <n>     : insertnp/INSERTION_OF_NT max allowed nucleotide insertion length in blastx validation is <n> [27]
  --xmaxdel <n>     : deletinp/DELETION_OF_NT max allowed nucleotide deletion length in blastx validation is <n> [27]
  --nmaxins <n>     : insertnn/INSERTION_OF_NT max allowed nucleotide (nt) insertion length in CDS nt alignment is <n> [27]
  --nmaxdel <n>     : deletinn/DELETION_OF_NT max allowed nucleotide (nt) deletion length in CDS nt alignment is <n> [27]
  --xlonescore <n>  : indfantp/INDEFINITE_ANNOTATION min score for a blastx hit not supported by CM analysis is <n> [80]
  --hlonescore <n>  : indfantp/INDEFINITE_ANNOTATION min score for a hmmer hit not supported by CM analysis is <n> [10]

options for controlling cmalign alignment stage:
  --mxsize <n> : set max allowed memory for cmalign to <n> Mb [16000]
  --tau <x>    : set the initial tau value for cmalign to <x> [0.001]
  --nofixedtau : do not fix the tau value when running cmalign, allow it to decrease if nec
  --nosub      : use alternative alignment strategy for truncated sequences
  --noglocal   : do not run cmalign in glocal mode (run in local mode)

options for controlling blastx protein validation stage:
  --xmatrix <s> : use the matrix <s> with blastx (e.g. BLOSUM45)
  --xdrop <n>   : set the xdrop value for blastx to <n> [25]
  --xnumali <n> : number of alignments to keep in blastx output and consider if --xlongest is <n> [20]
  --xlongest    : keep the longest blastx hit, not the highest scoring one

options for using hmmer instead of blastx for protein validation:
  --hmmer        : use hmmer for protein validation, not blastx
  --h_max        : use --max option with hmmsearch
  --h_minbit <x> : set minimum hmmsearch bit score threshold to <x> [-10]

options related to blastn-derived seeded alignment acceleration:
  -s               : use the max length ungapped region from blastn to seed the alignment
  --s_blastnws <n> : for -s, set blastn -word_size <n> to <n> [7]
  --s_blastnsc <x> : for -s, set blastn minimum HSP score to consider to <x> [50]
  --s_overhang <n> : for -s, set length of nt overhang for subseqs to align to <n> [100]

options related to replacing Ns with expected nucleotides:
  -r               : replace stretches of Ns with expected nts, where possible
  --r_minlen <n>   : minimum length subsequence to replace Ns in is <n> [5]
  --r_minfract <x> : minimum fraction of Ns in subseq to trigger replacement is <x> [0.5]
  --r_fetchr       : fetch features for output fastas from seqs w/Ns replaced, not originals
  --r_cdsmpr       : detect CDS and MP alerts in sequences w/Ns replaced, not originals
  --r_pvorig       : use original sequences for protein validation, not replaced seqs
  --r_prof         : use slower profile methods, not blastn, to identify Ns to replace

options related to parallelization on compute farm:
  -p             : parallelize cmsearch/cmalign on a compute farm
  -q <s>         : use qsub info file <s> instead of default
  --nkb <n>      : number of KB of sequence for each farm job is <n> [10]
  --wait <n>     : allow <n> wall-clock minutes for jobs on farm to finish, including queueing time [500]
  --errcheck     : consider any farm stderr output as indicating a job failure
  --maxnjobs <n> : set max number of jobs to submit to compute farm to <n> [2500]

options for skipping stages:
  --skip_align : skip the cmalign step, use results from an earlier run of the script
  --skip_pv    : do not perform blastx-based protein validation

optional output files:
  --out_stk     : output per-model full length stockholm alignments (.stk)
  --out_afa     : output per-model full length fasta alignments (.afa)
  --out_rpstk   : with -r, output stockholm alignments of seqs with Ns replaced
  --out_rpafa   : with -r, output fasta alignments of seqs with Ns replaced
  --out_nofs    : do not output frameshift stockholm alignment files
  --out_nofasta : do not output fasta files of features, or passing/failing seqs
  --out_debug   : dump voluminous info from various data structures to output files

other expert options:
  --execname <s> : define executable name of this script as <s>
  --alicheck     : for debugging, check aligned sequence vs input sequence for identity
  --noseqnamemax : do not enforce a maximum length of 50 for sequence names (GenBank max)
  --minbit <x>   : set minimum cmsearch bit score threshold to <x> [-10]
  --origfa       : do not copy fasta file prior to analysis, use original
  --msub <s>     : read model substitution file from <s>
  --xsub <s>     : read blastx db substitution file from <s>
End the interactive session:
[user@cn4471 ~]$ exit
salloc.exe: Relinquishing job allocation 46116226
[user@biowulf ~]$