EMBOSS: getorf

| getorf

## Function

Finds and extracts open reading frames (ORFs)

## Description

This program finds and outputs the sequences of open reading frames (ORFs) in one or more nucleotide sequences. An ORF may be defined as a region of a specified minimum size between two STOP codons, or between a START and a STOP codon. The ORFs can be output as the nucleotide sequence or as the protein translation. Optionally, the program will output the region around the START codon, the first STOP codon, or the final STOP codon of an ORF. The START and STOP codons are defined in a Genetic Code table; a suitable table can be selected for the organism you are investigating. The output is a sequence file containing predicted open reading frames longer than the minimum size, which defaults to 30 bases (i.e. 10 amino acids).

## Usage

Here is a sample session with getorf

    % getorf -minsize 300
    Finds and extracts open reading frames (ORFs)
    Input nucleotide sequence(s): tembl:v00294
    protein output sequence(s) [v00294.orf]:

Go to the input files for this example
Go to the output files for this example

## Command line arguments

       Standard (Mandatory) qualifiers:
      [-sequence]          seqall     Nucleotide sequence(s) filename and optional
                                      format, or reference (input USA)
      [-outseq]            seqoutall  [.] Protein sequence
                                      set(s) filename and optional format (output
                                      USA)

       Additional (Optional) qualifiers:
       -table              menu       [0] Code to use (Values: 0 (Standard); 1
                                      (Standard (with alternative initiation
                                      codons)); 2 (Vertebrate Mitochondrial); 3
                                      (Yeast Mitochondrial); 4 (Mold, Protozoan,
                                      Coelenterate Mitochondrial and
                                      Mycoplasma/Spiroplasma); 5 (Invertebrate
                                      Mitochondrial); 6 (Ciliate Macronuclear and
                                      Dasycladacean); 9 (Echinoderm
                                      Mitochondrial); 10 (Euplotid Nuclear); 11
                                      (Bacterial); 12 (Alternative Yeast Nuclear);
                                      13 (Ascidian Mitochondrial); 14 (Flatworm
                                      Mitochondrial); 15 (Blepharisma
                                      Macronuclear); 16 (Chlorophycean
                                      Mitochondrial); 21 (Trematode
                                      Mitochondrial); 22 (Scenedesmus obliquus);
                                      23 (Thraustochytrium Mitochondrial))
       -minsize            integer    [30] Minimum nucleotide size of ORF to
                                      report (Any integer value)
       -maxsize            integer    [1000000] Maximum nucleotide size of ORF to
                                      report (Any integer value)
       -find               menu       [0] This is a small menu of possible output
                                      options. The first four options are to
                                      select either the protein translation or the
                                      original nucleic acid sequence of the open
                                      reading frame. There are two possible
                                      definitions of an open reading frame: it can
                                      either be a region that is free of STOP
                                      codons or a region that begins with a START
                                      codon and ends with a STOP codon. The last
                                      three options are probably only of interest
                                      to people who wish to investigate the
                                      statistical properties of the regions around
                                      potential START or STOP codons. The last
                                      option assumes that ORF lengths are
                                      calculated between two STOP codons. (Values:
                                      0 (Translation of regions between STOP
                                      codons); 1 (Translation of regions between
                                      START and STOP codons); 2 (Nucleic sequences
                                      between STOP codons); 3 (Nucleic sequences
                                      between START and STOP codons); 4
                                      (Nucleotides flanking START codons); 5
                                      (Nucleotides flanking initial STOP codons);
                                      6 (Nucleotides flanking ending STOP codons))

       Advanced (Unprompted) qualifiers:
       -[no]methionine     boolean    [Y] START codons at the beginning of protein
                                      products will usually code for Methionine,
                                      despite what the codon will code for when it
                                      is internal to a protein. This qualifier
                                      sets all such START codons to code for
                                      Methionine by default.
       -circular           boolean    [N] Is the sequence circular
       -[no]reverse        boolean    [Y] Set this to be false if you do not wish
                                      to find ORFs in the reverse complement of
                                      the sequence.
       -flanking           integer    [100] If you have chosen one of the options
                                      of the type of sequence to find that gives
                                      the flanking sequence around a STOP or START
                                      codon, this allows you to set the number of
                                      nucleotides either side of that codon to
                                      output. If the region of flanking
                                      nucleotides crosses the start or end of the
                                      sequence, no output is given for this codon.
                                      (Any integer value)

       Associated qualifiers:

       "-sequence" associated qualifiers
       -sbegin1            integer    Start of each sequence to be used
       -send1              integer    End of each sequence to be used
       -sreverse1          boolean    Reverse (if DNA)
       -sask1              boolean    Ask for begin/end/reverse
       -snucleotide1       boolean    Sequence is nucleotide
       -sprotein1          boolean    Sequence is protein
       -slower1            boolean    Make lower case
       -supper1            boolean    Make upper case
       -sformat1           string     Input sequence format
       -sdbname1           string     Database name
       -sid1               string     Entryname
       -ufo1               string     UFO features
       -fformat1           string     Features format
       -fopenfile1         string     Features file name

       "-outseq" associated qualifiers
       -osformat2          string     Output seq format
       -osextension2       string     File name extension
       -osname2            string     Base file name
       -osdirectory2       string     Output directory
       -osdbname2          string     Database name to add
       -ossingle2          boolean    Separate file for each entry
       -oufo2              string     UFO features
       -offormat2          string     Features format
       -ofname2            string     Features file name
       -ofdirectory2       string     Output directory

       General qualifiers:
       -auto               boolean    Turn off prompts
       -stdout             boolean    Write first file to standard output
       -filter             boolean    Read first file from standard input, write
                                      first file to standard output
       -options            boolean    Prompt for standard and additional values
       -debug              boolean    Write debug output to program.dbg
       -verbose            boolean    Report some/full command line options
       -help               boolean    Report command line options. More
                                      information on associated and general
                                      qualifiers can be found with -help -verbose
       -warning            boolean    Report warnings
       -error              boolean    Report errors
       -fatal              boolean    Report fatal errors
       -die                boolean    Report dying program messages

Standard (Mandatory) qualifiers | Allowed values | Default
--- | --- | ---
[-sequence] (Parameter 1) | Nucleotide sequence(s) filename and optional format, or reference (input USA) | Readable sequence(s) | Required
[-outseq] (Parameter 2) | Protein sequence set(s) filename and optional format (output USA) | Writeable sequence(s) | <*>.format
Additional (Optional) qualifiers | Allowed values | Default
-table | Code to use |
0 | (Standard)
1 | (Standard (with alternative initiation codons))
2 | (Vertebrate Mitochondrial)
3 | (Yeast Mitochondrial)
4 | (Mold, Protozoan, Coelenterate Mitochondrial and Mycoplasma/Spiroplasma)
5 | (Invertebrate Mitochondrial)
6 | (Ciliate Macronuclear and Dasycladacean)
9 | (Echinoderm Mitochondrial)
10 | (Euplotid Nuclear)
11 | (Bacterial)
12 | (Alternative Yeast Nuclear)
13 | (Ascidian Mitochondrial)
14 | (Flatworm Mitochondrial)
15 | (Blepharisma Macronuclear)
16 | (Chlorophycean Mitochondrial)
21 | (Trematode Mitochondrial)
22 | (Scenedesmus obliquus)
23 | (Thraustochytrium Mitochondrial)
| 0
-minsize | Minimum nucleotide size of ORF to report | Any integer value | 30
-maxsize | Maximum nucleotide size of ORF to report | Any integer value | 1000000
-find | This is a small menu of possible output options. The first four options are to select either the protein translation or the original nucleic acid sequence of the open reading frame. There are two possible definitions of an open reading frame: it can either be a region that is free of STOP codons or a region that begins with a START codon and ends with a STOP codon. The last three options are probably only of interest to people who wish to investigate the statistical properties of the regions around potential START or STOP codons. The last option assumes that ORF lengths are calculated between two STOP codons. |
0 | (Translation of regions between STOP codons)
1 | (Translation of regions between START and STOP codons)
2 | (Nucleic sequences between STOP codons)
3 | (Nucleic sequences between START and STOP codons)
4 | (Nucleotides flanking START codons)
5 | (Nucleotides flanking initial STOP codons)
6 | (Nucleotides flanking ending STOP codons)
| 0
Advanced (Unprompted) qualifiers | Allowed values | Default
-[no]methionine | START codons at the beginning of protein products will usually code for Methionine, despite what the codon will code for when it is internal to a protein. This qualifier sets all such START codons to code for Methionine by default. | Boolean value Yes/No | Yes
-circular | Is the sequence circular | Boolean value Yes/No | No
-[no]reverse | Set this to be false if you do not wish to find ORFs in the reverse complement of the sequence. | Boolean value Yes/No | Yes
-flanking | If you have chosen one of the options of the type of sequence to find that gives the flanking sequence around a STOP or START codon, this allows you to set the number of nucleotides either side of that codon to output. If the region of flanking nucleotides crosses the start or end of the sequence, no output is given for this codon. | Any integer value | 100

## Input file format

getorf reads any nucleic acid sequence USA.

### Input files for usage example

'tembl:v00294' is a sequence entry in the example nucleic acid database 'tembl'

### Database entry: tembl:v00294

    ID   V00294; SV 1; linear; genomic DNA; STD; PRO; 1113 BP.
    XX
    AC   V00294;
    XX
    DT   09-JUN-1982 (Rel. 01, Created)
    DT   10-FEB-1999 (Rel. 58, Last updated, Version 2)
    XX
    DE   E. coli laci gene (codes for the lac repressor).
    XX
    KW   DNA binding protein; repressor.
    XX
    OS   Escherichia coli
    OC   Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales;
    OC   Enterobacteriaceae; Escherichia.
    XX
    RN   [1]
    RP   1-1113
    RX   DOI; 10.1038/274765a0.
    RX   PUBMED; 355891.
    RA   Farabaugh P.J.;
    RT   "Sequence of the lacI gene";
    RL   Nature 274(5673):765-769(1978).
    XX
    CC   KST ECO.LACI
    XX
    FH   Key             Location/Qualifiers
    FH
    FT   source          1..1113
    FT                   /organism="Escherichia coli"
    FT                   /mol_type="genomic DNA"
    FT                   /db_xref="taxon:562"
    FT   CDS             31..1113
    FT                   /transl_table=11
    FT                   /note="reading frame"
    FT                   /db_xref="GOA:P03023"
    FT                   /db_xref="PDB:1CJG"
    FT                   /db_xref="PDB:1EFA"
    FT                   /db_xref="PDB:1JWL"
    FT                   /db_xref="PDB:1JYE"
    FT                   /db_xref="PDB:1JYF"
    FT                   /db_xref="PDB:1L1M"
    FT                   /db_xref="PDB:1LBG"
    FT                   /db_xref="PDB:1LBH"
    FT                   /db_xref="PDB:1LBI"
    FT                   /db_xref="PDB:1LCC"
    FT                   /db_xref="PDB:1LCD"
    FT                   /db_xref="PDB:1LQC"
    FT                   /db_xref="PDB:1LTP"
    FT                   /db_xref="PDB:1TLF"
    FT                   /db_xref="PDB:2BJC"
    FT                   /db_xref="UniProtKB/Swiss-Prot:P03023"
    FT                   /protein_id="CAA23569.1"
    FT                   /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
    FT                   NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
    FT                   EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
    FT                   EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
    FT                   MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
    FT                   YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
    FT                   ALADSLMQLARQVSRLESGQ"
    XX
    SQ   Sequence 1113 BP; 249 A; 304 C; 322 G; 238 T; 0 other;
         ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca        60
         gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt       120
         tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac       180
         cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt       240
         ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg       300
         ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg       360
         gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac       420
         caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc       480
         tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc       540
         gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt       600
         tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt       660
         cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg       720
         caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg       780
         ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta       840
         gtgggatacg acgataccga agacagctca tgttatatcc cgccgtcaac caccatcaaa       900
         caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc       960
         caggcggtga agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg      1020
         gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca      1080
         cgacaggttt cccgactgga aagcgggcag tga                                   1113
    //

## Output file format

The output is a sequence file containing predicted open reading frames longer than the minimum size, which defaults to 30 bases (i.e. 10 amino acids).

### Output files for usage example

### File: v00294.orf

    >V00294_1 [735 - 1112] E. coli laci gene (codes for the lac repressor).
    GHRSHCDAGCQRSDGAGRNARHYRVRAARWCGYLGSGIRRYRRQLMLYPAVNHHQTGFSP
    AGANQRGPLAATLSGPGGEGQSAVARLTGEKKNHPGAQYANRLSPRVGRFINAAGTTGFP
    TGKRAV
    >V00294_2 [1 - 1110] E. coli laci gene (codes for the lac repressor).
    PEESQFRVVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPN
    RVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAA
    VHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLG
    VEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTM
    QMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPSTTIK
    QDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLA
    RQVSRLESGQ*
    >V00294_3 [465 - 49] (REVERSE SENSE) E. coli laci gene (codes for the lac repressor).
    RRNISAGSFHSNGILVIQRIVNDQPTDALREKIVHRRFTGFDAASFYHRHHHAGTQLIGA
    RFNRRDNLRRRVQGQTGGGNANQQRLFARQLLCHAVGNVIQLRHRRFHFFPRFRRNVAGL
    VHHAGNGLIRDTGILCDIV

The name of the ORF sequences is constructed from the name of the input sequence with an underscore character ('_') and a unique ordinal number of the ORF found appended. The description of the output ORF sequence is constructed from the description of the input sequence with the start and end positions of the ORF prepended.

The unique number appended to the name is simply used to create new unique sequence names, it does not imply any further information indicating any order, positioning or sense-strand of the ORFs.

If the ORF has been found in the reverse sense, then the start position will be smaller than the end position. The numbering uses the forward-sense positions, but read in the reverse sense. For example, >V00294_3 [465 - 49] in the output above is a reverse-sense ORF running from position 465 to 49. The description will also contain '(REVERSE SENSE)'.

If the sequence has been specified as a circular genome (using the command-line switch '-circular'), then ORFs can potentially continue past the 'end' of the input sequence (the breakpoint of the circular genome) and into the 'start' of the sequence again. This is dealt with by appending the sequence to itself three times and reporting long ORFs that are found in this extended sequence. Any ORF that is longer that three times the sequence length (i.e one that continues without hitting a STOP at any point in the genome) will be reported as being a maximum of three times the length of the input sequence. Note that the end position of an ORF in circular genomes can be apparently longer than the input sequence if the ORF crosses the breakpoint. If the ORF crosses the breakpoint, then the text '(ORF crosses the breakpoint)' will be added to the description of the output sequence.

## Data files

The START and STOP codons used by getorf are defined in the Genetic Code data files. By default, Genetic Code file EGC.0 is used.

The default file EGC.0 is the 'Standard Code' with the rarely used alternate START codons omitted, it only has the normal 'AUG' START codon. The 'Standard Code' with the rarely used alternate START codons included is Genetic Code file EGC.1.

It is expected that user will sometimes wish to customise a Genetic Code file. To do this, use the program embossdata.

EMBOSS data files are distributed with the application and stored in the standard EMBOSS data directory, which is defined by the EMBOSS environment variable EMBOSS_DATA.

To see the available EMBOSS data files, run:

    % embossdata -showall

To fetch one of the data files (for example 'Exxx.dat') into your current directory for you to inspect or modify, run:

    % embossdata -fetch -file Exxx.dat

Users can provide their own data files in their own directories. Project specific files can be put in the current directory, or for tidier directory listings in a subdirectory called ".embossdata". Files for all EMBOSS runs can be put in the user's home directory, or again in a subdirectory called ".embossdata".

The directories are searched in the following order:

- . (your current directory)
- .embossdata (under your current directory)
- ~/ (your home directory)
- ~/.embossdata

The Genetic Code data files are based on the NCBI genetic code tables. Their names and descriptions are:

EGC.0
  Standard (Differs from GC.1 in that it only has initiation site 'AUG')
EGC.1
  Standard
EGC.2
  Vertebrate Mitochodrial
EGC.3
  Yeast Mitochondrial
EGC.4
  Mold, Protozoan, Coelenterate Mitochondrial and Mycoplasma/Spiroplasma
EGC.5
  Invertebrate Mitochondrial
EGC.6
  Ciliate Macronuclear and Dasycladacean
EGC.9
  Echinoderm Mitochondrial
EGC.10
  Euplotid Nuclear
EGC.11
  Bacterial
EGC.12
  Alternative Yeast Nuclear
EGC.13
  Ascidian Mitochondrial
EGC.14
  Flatworm Mitochondrial
EGC.15
  Blepharisma Macronuclear
EGC.16
  Chlorophycean Mitochondrial
EGC.21
  Trematode Mitochondrial
EGC.22
  Scenedesmus obliquus
EGC.23
  Thraustochytrium Mitochondrial

The format of these files is very simple.

It consists of several lines of optional comments, each starting with a '#' character.

These are followed the line: 'Genetic Code [n]', where 'n' is the number of the genetic code file.

This is followed by the description of the code and then by four lines giving the IUPAC one-letter code of the translated amino acid, the start codons (indicdated by an 'M') and the three bases of the codon, lined up one on top of the other.

For example:

    ------------------------------------------------------------------------------
    # Genetic Code Table
    #
    # Obtained from: http://www.ncbi.nlm.nih.gov/collab/FT/genetic_codes.html
    # and: http://www3.ncbi.nlm.nih.gov/htbin-post/Taxonomy/wprintgc?mode=c
    #
    # Differs from Genetic Code [1] only in that the initiation sites have been
    # changed to only 'AUG'

    Genetic Code [0]
    Standard

    AAs  =   FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG
    Starts = -----------------------------------M----------------------------
    Base1  = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG
    Base2  = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG
    Base3  = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG
    ------------------------------------------------------------------------------

## Notes

There are two common definitions of an open reading frame: it can either be a region that is free of STOP codons or a region that begins with a START codon and ends with a STOP codon.

## References

None.

## Warnings

None.

## Diagnostic Error Messages

None.

## Exit status

It always exits with status 0.

## Known bugs

'-sbegin' and -send' do not work with this program.

## See also

Program name | Description
--- | ---
marscan | Finds matrix/scaffold recognition (MRS) signatures in DNA sequences
plotorf | Plot potential open reading frames in a nucleotide sequence
showorf | Display a nucleotide sequence and translation in pretty format
sixpack | Display a DNA sequence with 6-frame translation and ORFs
syco | Draw synonymous codon usage statictic plot for a nucleotide sequence
tcode | Identify protein-coding regions using Fickett TESTCODE statistic
wobble | Plot third base position variability in a nucleotide sequence

- checktrans - Reports STOP codons and ORF statistics of a protein sequence

## Author(s)

Gary Williams (gwilliam © rfcgr.mrc.ac.uk)
MRC Rosalind Franklin Centre for Genomics Research Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SB, UK

## History

2000 - written - Gary Williams

November 2002 - added indication of reverse sense ORFs

November 2002 - added indication of ORFs that cross the breakpoint at position 1 in circular genomes.

## Target users

This program is intended to be used by everyone and everything, from naive users to embedded scripts.

## Comments

None