The ORFs can be defined as regions of a specified minimum size between STOP codons or between START and STOP codons.
The ORFs can be output as the nucleotide sequence or as the translation.
The program can also output the region around the START or the initial STOP codon or the ending STOP codons of an ORF for those doing analysis of the properties of these regions.
The START and STOP codons are defined in the Genetic Code tables. A suitable Genetic Code table can be selected for the organism you are investigating.
% getorf -minsize 300 Finds and extracts open reading frames (ORFs) Input sequence(s): tembl:eclaci Output sequence [eclaci.orf]:
Go to the input files for this example
Go to the output files for this example
Standard (Mandatory) qualifiers: [-sequence] seqall Sequence database USA [-outseq] seqoutall Output sequence(s) USA Additional (Optional) qualifiers: -table menu Code to use -minsize integer Minimum nucleotide size of ORF to report -maxsize integer Maximum nucleotide size of ORF to report -find menu 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. Advanced (Unprompted) qualifiers: -[no]methionine boolean 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 Is the sequence circular -[no]reverse boolean Set this to be false if you do not wish to find ORFs in the reverse complement of the sequence. -flanking integer 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. 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 standard output -filter boolean Read standard input, write 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 deaths
|Standard (Mandatory) qualifiers||Allowed values||Default|
|Sequence database USA||Readable sequence(s)||Required|
|Output sequence(s) USA||Writeable sequence(s)||<sequence>.format|
|Additional (Optional) qualifiers||Allowed values||Default|
|-table||Code to use||
|-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.||
|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|
ID ECLACI standard; DNA; PRO; 1113 BP. XX AC V00294; XX SV V00294.1 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; gamma subdivision; Enterobacteriaceae; OC Escherichia. XX RN  RP 1-1113 RX MEDLINE; 78246991. RA Farabaugh P.J.; RT "Sequence of the lacI gene"; RL Nature 274:765-769(1978). XX DR SWISS-PROT; P03023; LACI_ECOLI. XX CC KST ECO.LACI XX FH Key Location/Qualifiers FH FT source 1..1113 FT /db_xref="taxon:562" FT /organism="Escherichia coli" FT CDS 31..1113 FT /db_xref="SWISS-PROT:P03023" FT /note="reading frame" FT /transl_table=11 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 //
>ECLACI_1 [735 - 1112] E. coli laci gene (codes for the lac repressor). GHRSHCDAGCQRSDGAGRNARHYRVRAARWCGYLGSGIRRYRRQLMLYPAVNHHQTGFSP AGANQRGPLAATLSGPGGEGQSAVARLTGEKKNHPGAQYANRLSPRVGRFINAAGTTGFP TGKRAV >ECLACI_2 [1 - 1110] E. coli laci gene (codes for the lac repressor). PEESQFRVVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPN RVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGVEACKAA VHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSHEDGTRLG VEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTM QMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPSTTIK QDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLA RQVSRLESGQ* >ECLACI_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, >ECLACI_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.
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:
The Genetic Code data files are based on the NCBI genetic code tables. Their names and descriptions are:
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.
------------------------------------------------------------------------------ # 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  only in that the initiation sites have been # changed to only 'AUG' Genetic Code  Standard AAs = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG Starts = -----------------------------------M---------------------------- Base1 = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG Base2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG Base3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG ------------------------------------------------------------------------------
|marscan||Finds MAR/SAR sites in nucleic sequences|
|plotorf||Plot potential open reading frames|
|showorf||Pretty output of DNA translations|
|sixpack||Display a DNA sequence with 6-frame translation and ORFs|
|syco||Synonymous codon usage Gribskov statistic plot|
|tcode||Fickett TESTCODE statistic to identify protein-coding DNA|
|wobble||Wobble base plot|
November 2002 - added indication of reverse sense ORFs
November 2002 - added indication of ORFs that cross the breakpoint at position 1 in circular genomes.