StORF-Reporter has now been published in NAR: https://doi.org/10.1093/nar/gkad814
StORF-Reporter, a toolkit that returns missed CDS genes from the Unannotated Regions (URs) of prokaryotic genomes.
This will also install the python-standard library numpy (>=1.22.0,<1.24.0) and Pyrodigal (https://github.com/althonos/pyrodigal).
Consider using '--no-cache-dir' with pip to ensure the download of the newest version of StORF-Reporter.
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Supplement a current annotation from a tool such as Prokka or Bakta. A new GFF file will be created compatible with downstream pangenome analysis tools such as Roary and Panaroo.
For use on a single Prokka/Bakta output directory - Will also create a new fasta file with Prokka/Bakta genes and StORF sequences.
StORF-Reporter -anno Prokka Out_Dir -p .../Test_Datasets/Prokka_E-coli/For use on multiple Prokka/Bakta output directies - Will also create a new fasta file with Prokka/Bakta genes and StORF sequences.
StORF-Reporter -anno Prokka Multiple_Out_Dirs -p ../Test_Datasets/Multi_Prokka_OutsStORF-Reporter -anno Prokka Multiple_GFFs -p .../Test_Datasets/Prokka_Outputs/For use on a GFF file from a CDS prediction tool such as Prodigal - Provide a GFF file and StORF-Reporter will find the matching .fa/.fasta/.fna (must have the same name).
StORF-Reporter -anno Feature_Types Single_Genome -p .../Test_Datasets/Matching_GFF_FASTA/Myco.gffFor use on a directory containing multiple GFF files from a CDS prediction tool such as Prodigal - StORF-Reporter will find the matching .fa/.fasta/.fna (must have the same name).
StORF-Reporter -anno Feature_Types Multiple_Genomes -p .../Test_Datasets/Matching_GFF_FASTA/StORF-Reporter -anno Feature_Types Multiple_Combined_GFFs -p .../Test_Datasets/Combined_GFFs/To perform a fresh end-to-end annotation of a genome without an annotation, StORF-Reporter will use Pyrodigal to predict CDS genes and then supplement with StORFs.
StORF-Reporter -anno Pyrodigal Single_FASTA -p .../Test_Datasets/Pyrodigal/E-coli.faStORF-Reporter -anno Ensembl Single_Genome -p .../Test_Datasets/Matching_GFF_FASTA/E-coli.gffusage: StORF_Reporter.py [-h]
[-anno [{Prokka,Bakta,Out_Dir,Multiple_Out_Dirs,Single_GFF,Multiple_GFFs,Ensembl,Feature_Types,Single_Genome,Multiple_Genomes,Single_Combined_GFF,Multiple_Combined_GFFs,Pyrodigal,Single_FASTA,Multiple_FASTA} ...]]
[-p PATH] [-af ALT_FILENAME] [-oname O_NAME]
[-odir O_DIR] [-sout {True,False}] [-lw {True,False}]
[-aa {True,False}] [-gz {True,False}]
[-py_train [{longest,individual,meta}]]
[-py_fasta {True,False}] [-py_unstorfed {True,False}]
[-gene_ident GENE_IDENT] [-min_len MINLEN]
[-max_len MAXLEN] [-ex_len EXLEN]
[-spos {True,False}] [-rs {True,False}]
[-con_storfs {True,False}] [-con_only {True,False}]
[-ps {True,False}] [-wc {True,False}]
[-short_storfs {False,Nolap,Olap}]
[-short_storfs_only {True,False}] [-minorf MIN_ORF]
[-maxorf MAX_ORF] [-codons STOP_CODONS]
[-olap_filt [{none,single-strand,both-strand}]]
[-start_filt {True,False}] [-so [{start_pos,strand}]]
[-f_type [{StORF,CDS,ORF}]]
[-non_standard NON_STANDARD] [-olap OVERLAP_NT]
[-ao ALLOWED_OVERLAP] [-overwrite {True,False}]
[-verbose {True,False}] [-v]
StORF-Reporter v1.4.3: StORF-Reporter Run Parameters.
Required Options:
-anno [{Prokka,Bakta,Out_Dir,Multiple_Out_Dirs,Single_GFF,Multiple_GFFs,Ensembl,Feature_Types,Single_Genome,Multiple_Genomes,Single_Combined_GFF,Multiple_Combined_GFFs,Pyrodigal,Single_FASTA,Multiple_FASTA} ...]
Select Annotation and Input options for one of the 3 options listed below
### Prokka/Bakta Annotation Option 1:
Prokka = Report StORFs for a Prokka annotation;
Bakta = Report StORFs for a Bakta annotation;
--- Prokka/Bakta Input Options:
Out_Dir = To provide the output directory of either a Prokka or Bakta run (will produce a new GFF and FASTA file containing original and extended annotations);
Multiple_Out_Dirs = To provide a directory containing multiple Prokka/Bakta standard output directories - Will run on each sequentially;
Single_GFF = To provide a single Prokka or Bakta GFF - searches for accompanying ".fna" file (will provide a new extended GFF);
Multiple_GFFs = To provide a directory containing multiple Prokka or Bakta GFF files - searches for accompanying ".fna" files (will provide a new extended GFF);
### Standard GFF Annotation Option 2:
Ensembl = Report StORFs for an Ensembl Bacteria annotation (ID=gene);
Feature_Types = Used in conjunction with -gene_ident to define features such as CDS,rRNA,tRNA for UR extraction (default CDS);
--- Standard GFF Input Options:
Single_Genome = To provide a single Genome - accompanying FASTA must share same name as given gff file (can be .fna, .fa or .fasta);
Multiple_Genomes = To provide a directory containing multiple accompanying GFF and FASTA files - files must share the same name (fasta can be .fna, .fa or .fasta);
Single_Combined_GFF = To provide a GFF file with embedded FASTA at the bottom;
Multiple_Combined_GFFs = To provide a directory containing multiple GFF files with embedded FASTA at the bottom;
### Complete Annotation Option 3:
Pyrodigal = Run Pyrodigal then Report StORFs (provide path to single FASTA or directory of multiple FASTA files ;
--- Complete Annotation Input Options:
Single_FASTA = To provide a single FASTA file;
Multiple_FASTA = To provide a directory containing multiple FASTA files (will detect .fna,.fa,.fasta);
-p PATH Provide input file or directory path
StORF-Reporter Options:
-af ALT_FILENAME Default - Prokka/Bakta output directory share the same
prefix with their gff/fna files - Use this option when
Prokka/Bakta output directory name is different from
the gff/fna files within and StORF-Reporter will
search for the gff/fna with the given prefix
(MyProkkaDir/"altname".gff) - Does not work with
"Multiple_Out_Dirs" option
-oname O_NAME Default - Appends '_StORF-Reporter_Extended' to end of
input filename - Takes the directory name of
Prokka/Bakta output if given as input or the input for
-af if given - Multiple_* runs will be numbered
-odir O_DIR Default - Same directory as input
-sout {True,False} Default - False: Print out StORF sequences separately
from Prokka/Bakta annotations
-lw {True,False} Default - True: Line wrap FASTA sequence output at 60
chars
-aa {True,False} Default - False: Report StORFs as amino acid sequences
-gz {True,False} Default - False: Output as .gz
Pyrodigal Options:
-py_train [{longest,individual,meta}]
Default - longest: Type of model training to be done
for Pyrodigal CDS prediction: Options: longest =
Trains on longest contig; individual = Trains on each
contig separately - runs in meta mode if contig is <
20KB; meta = Runs in meta mode for all sequences
-py_fasta {True,False}
Default - False: Output Pyrodigal+StORF predictions in
FASTA format
-py_unstorfed {True,False}
Default - False: Provide GFF containing original
Pyrodigal predictions
UR-Extractor Options:
-gene_ident GENE_IDENT
Default: "CDS". Specifies feature types to exclude
from Unannotated rRegion extraction. Provide a comma-
separated list of feature types, e.g., "misc_RNA,gene,
mRNA,CDS,rRNA,tRNA,tmRNA,CRISPR,ncRNA,regulatory_regio
n,oriC,pseudo", to identify annotated regions. - To be
used with "-anno Feature_Types" - "-gene_ident Prokka"
will select "most" features present in Prokka/Bakta
annotations- Providing "ID=gene" will check the
attribute column for features assigned as genes
(compatible with Ensembl annotations). All regions
without these feature types will be extracted as
unannotated.
-min_len MINLEN Default - 30: Minimum UR Length
-max_len MAXLEN Default - 100,000: Maximum UR Length
-ex_len EXLEN Default - 50: UR Extension Length
StORF-Finder Options:
-spos {True,False} Default - False: Output StORF sequences and GFF
positions inclusive of first stop codon -This can
break some downstream tools if changed to True.
-rs {True,False} Default - True: Remove stop "*" from StORF amino acid
sequences
-con_storfs {True,False}
Default - False: Output Consecutive StORFs
-con_only {True,False}
Default - False: Only output Consecutive StORFs
-ps {True,False} Default - False: Partial StORFs reported
-wc {True,False} Default - False: StORFs reported across entire
sequence
-short_storfs {False,Nolap,Olap}
Default - False: Run StORF-Finder in "Short-StORF"
mode. Will only return StORFs between 30 and 120 nt
that do not overlap longer StORFs - Only works with
StORFs for now. "Nolap" will filter Short-StORFs which
areoverlapped by StORFs and Olap will report Short-
StORFs which do overlap StORFs. Overlap is defined by
"-olap".
-short_storfs_only {True,False}
Default - True. Only report Short-StORFs?
-minorf MIN_ORF Default - 99: Minimum StORF size in nt
-maxorf MAX_ORF Default - 60kb: Maximum StORF size in nt
-codons STOP_CODONS Default - ('TAG,TGA,TAA'): List Stop Codons to use
-olap_filt [{none,single-strand,both-strand}]
Default - "both-strand": Filtering level "none" is not
recommended, "single-strand" for single strand
filtering and both-strand for both-strand longest-
first tiling
-start_filt {True,False}
Default - False: Filter out StORFs without at least
one of the 3 common start codons (best used for short-
storfs).
-so [{start_pos,strand}]
Default - Start Position: How should StORFs be ordered
when >1 reported in a single UR.
-f_type [{StORF,CDS,ORF}]
Default - "CDS": Which GFF feature type for StORFs to
be reported as in GFF - "CDS" is probably needed for
use in tools such as Roary and Panaroo
-non_standard NON_STANDARD
Default - 0.20: Reject StORFs with >=20% non-standard
nucleotides (A,T,G,C) - Provide % as decimal
-olap OVERLAP_NT Default - 50: Maximum number of nt of a StORF which
can overlap another StORF.
-ao ALLOWED_OVERLAP Default - 50 nt: Maximum overlap between a StORF and
an original gene.
Misc:
-overwrite {True,False}
Default - False: Overwrite StORF-Reporter output if
already present
-verbose {True,False}
Default - False: Print out runtime messages
-v Print out version number and exit
###################################
UR-Extractor -f .../Test_Datasets/Matching_GFF_FASTA/E-coli.fa -gff .../Test_Datasets/Matching_GFF_FASTA/E-coli.gffusage: UR_Extractor.py [-h] -gff GFF [-f FASTA] [-ident IDENT]
[-min_len MINLEN] [-max_len MAXLEN] [-ex_len EXLEN]
[-gene_ident GENE_IDENT] [-oname O_NAME] [-odir O_DIR]
[-gz {True,False}] [-verbose {True,False}] [-v]
StORF-Reporter v1.4.3: UR-Extractor Run Parameters.
Required Arguments:
-gff GFF GFF file containing genome annotation
Optional Arguments:
-f FASTA Accompanying FASTA file if GFF file does not contain
sequence data
-ident IDENT Identifier given for Unannotated Region output
sequences - Do not modify if output is to be used by
StORF-Finder: Default "Sequence-ID"_UR
-min_len MINLEN Minimum UR Length: Default 30
-max_len MAXLEN Maximum UR Length: Default 100,000
-ex_len EXLEN UR Extension Length on 5' and 3': Default 50
-gene_ident GENE_IDENT
Default: "CDS". Specifies feature types to exclude
from Unannotated rRegion extraction. Provide a comma-
separated list of feature types, e.g., "misc_RNA,gene,
mRNA,CDS,rRNA,tRNA,tmRNA,CRISPR,ncRNA,regulatory_regio
n,oriC,pseudo", to identify annotated regions.
"-gene_ident Prokka" will select "most" features
present in Prokka/Bakta annotations- Providing
"ID=gene" will check the attribute column for features
assigned as genes (compatible with Ensembl
annotations). All regions without these feature types
will be extracted as unannotated.
Output:
-oname O_NAME Default - Appends '_UR' to end of input GFF filename
-odir O_DIR Default - Same directory as input GFF
-gz {True,False} Default - False: Output as .gz
Misc:
-verbose {True,False}
Default - False: Print out runtime messages
-v Default - False: Print out version number and exit
StORF-Finder -f .../Test_Datasets/Matching_GFF_FASTA/E-coli_UR.fa usage: StORF_Finder.py [-h] -f FASTA [-ua {True,False}] [-wc {True,False}]
[-ps {True,False}]
[-olap_filt [{none,single-strand,both-strand}]]
[-start_filt {True,False}] [-con_storfs {True,False}]
[-con_only {True,False}]
[-short_storfs {False,Nolap,Olap}]
[-short_storfs_only {True,False}]
[-f_type [{StORF,CDS,ORF}]] [-minorf MIN_ORF]
[-maxorf MAX_ORF] [-codons STOP_CODONS]
[-non_standard NON_STANDARD] [-olap OVERLAP_NT]
[-s SUFFIX] [-so [{start_pos,strand}]] [-oname O_NAME]
[-odir O_DIR] [-gff {True,False}] [-aa {True,False}]
[-aa_only {True,False}] [-lw {True,False}]
[-spos {True,False}] [-stop_ident {True,False}]
[-gff_fasta {True,False}] [-gz {True,False}]
[-verbose {True,False}] [-v]
StORF-Reporter v1.4.3: StORF-Finder Run Parameters.
Required Arguments:
-f FASTA Input FASTA File - (UR_Extractor output)
Optional Arguments:
-ua {True,False} Default - Treat input as Unannotated: Use "-ua False"
for standard fasta
-wc {True,False} Default - False: StORFs reported across entire
sequence
-ps {True,False} Default - False: Partial StORFs reported
-olap_filt [{none,single-strand,both-strand}]
Default - "both-strand": Filtering level "none" is not
recommended, "single-strand" for single strand
filtering and both-strand for both-strand longest-
first tiling
-start_filt {True,False}
Default - False: Filter out StORFs without at least
one of the 3 common start codons (best used for short-
storfs).
-con_storfs {True,False}
Default - False: Output Consecutive StORFs
-con_only {True,False}
Default - False: Only output Consecutive StORFs
-short_storfs {False,Nolap,Olap}
Default - False: Run StORF-Finder in "Short-StORF"
mode. Will only return StORFs between 30 and 120 nt
that do not overlap longer StORFs - Only works with
StORFs for now. "Nolap" will filter Short-StORFs which
areoverlapped by StORFs and Olap will report Short-
StORFs which do overlap StORFs. Overlap is defined by
"-olap".
-short_storfs_only {True,False}
Default - True. Only report Short-StORFs?
-f_type [{StORF,CDS,ORF}]
Default - "StORF": Which GFF feature type for StORFs
to be reported as in GFF
-minorf MIN_ORF Default - 99: Minimum StORF size in nt
-maxorf MAX_ORF Default - 60kb: Maximum StORF size in nt
-codons STOP_CODONS Default - ('TAG,TGA,TAA'): List Stop Codons to use
-non_standard NON_STANDARD
Default - 0.20: Reject StORFs with >=20% non-standard
nucleotides (A,T,G,C) - Provide % as decimal
-olap OVERLAP_NT Default - 50: Maximum number of nt of a StORF which
can overlap another StORF.
-s SUFFIX Default - Do not append suffix to genome ID
-so [{start_pos,strand}]
Default - Start Position: How should StORFs be ordered
when >1 reported in a single UR.
Output:
-oname O_NAME Default - Appends '_StORF-Finder' to end of input
FASTA filename
-odir O_DIR Default - Same directory as input FASTA
-gff {True,False} Default - True: Output a GFF file
-aa {True,False} Default - False: Report StORFs as amino acid sequences
-aa_only {True,False}
Default - False: Only output Amino Acid Fasta
-lw {True,False} Default - True: Line wrap FASTA sequence output at 60
chars
-spos {True,False} Default - False: Output StORF sequences and GFF
positions inclusive of first stop codon -This can
break some downstream tools if changed to True.
-stop_ident {True,False}
Default - True: Identify Stop Codon positions with '*'
-gff_fasta {True,False}
Default - False: Report all gene sequences (nt) at the
bottom of GFF files in Prokka output mode
-gz {True,False} Default - False: Output as .gz
Misc:
-verbose {True,False}
Default - False: Print out runtime messages
-v Default - False: Print out version number and exit
Subpackage to extract sequences reported by StORF-Reporter from a genome annotation.
StORF-Extractor -storf_input Combined -p .../Test_Datasets/Combined_GFFs/E-coli_Combined_StORF-Reporter_Extended.gff usage: StORF_Extractor.py [-h] [-storf_input {Combined,Separate}] [-p PATH] [-gff_out {True,False}] [-oname O_NAME] [-odir O_DIR] [-gz {True,False}] [-verbose {True,False}] [-v]
StORF-Reporter v1.4.3: StORF-Extractor Run Parameters.
Required Arguments:
-storf_input {Combined,Separate}
Are StORFs to be extracted from Combined GFF/FASTA or Separate GFF/FASTA files?
-p PATH Provide input file or directory path
Output:
-gff_out {True,False}
Default - False: Output StORFs in GFF format
-oname O_NAME Default - Appends '_Extracted_StORFs' to end of input GFF filename
-odir O_DIR Default - Same directory as input FASTA
-gz {True,False} Default - False: Output as .gz
Misc:
-verbose {True,False}
Default - False: Print out runtime messages
-v Default - False: Print out version number and exit
Subpackage to remove sequences reported by StORF-Reporter without a Blast/Diamond hit (any alignment in BLAST 6 format).
StORF-Remover -gff .../Test_Datasets/StORF_Extractor_And_Remover/Myco_UR_StORF-R.gff -blast .../Test_Datasets/StORF_Extractor_And_Remover/Myco_URs_StORFs_aa_Swiss.tab usage: StORF_Remover.py [-h] [-gff GFF] [-blast BLAST] [-min_score MINSCORE] [-oname O_NAME] [-odir O_DIR] [-gz {True,False}]
[-verbose {True,False}] [-v]
StORF-Reporter v1.4.1: UR-Remover Run Parameters.
Required Arguments:
-gff GFF GFF annotation file for the FASTA
-blast BLAST BLAST format 6 annotation file
Optional Arguments:
-min_score MINSCORE Minimum BitScore to keep StORF: Default 30
Output:
-oname O_NAME Default - Appends '_UR' to end of input GFF filename
-odir O_DIR Default - Same directory as input GFF
-gz {True,False} Default - False: Output as .gz
Misc:
-verbose {True,False}
Default - False: Print out runtime messages
-v Default - False: Print out version number and exit