EMBOSS: tcode

| tcode

## Function

Identify protein-coding regions using Fickett TESTCODE statistic

## Description

tcode identifies protein-coding regions in one or more DNA sequences using the fickett TESTCODE statistic. This is based on simple and universal differences between protein-coding and noncoding DNA. The TESTCODE statistic is calculated for windows of a specified size over each input sequence. The results can be output as a standard EMBOSS report file or displayed graphically.

The output reports each window as "Coding", "Noncoding" or "No opinion". Entries marked "No opinion" have a TESTCODE value that falls between the maximum and minimum values required to report a region as noncoding or coding. For the graphical plot, all points above a green horizontal line are determined to be coding regions. Those below a red line are determined to be noncoding. Points between the red and green lines are "no opinion" ones.

## Biological Relevance

The statistic reflects the fact that codons are used with unequal frequency and that oligonucleotides and nucleotides tend to be repeated with a periodicity of three.

This application can assist in determining the probability of a region of nucleic sequence encoding a functional protein.

## Algorithm

The Fickett (1982) algorithm is used (1).

A window of at least 200 bases is moved over the sequence in steps of 3 bases

Let:

      A1 = Number of A's in positions 1,4,7 ...
      A2 = Number of A's in positions 2,5,8 ...
      A3 = Number of A's in positions 3,6,9 ...

A position value is determined that reflects the degree to which each base is favoured in one codon position over another, i.e.

      Apos = MAX(A1,A2,A3) / MIN(A1,A2,A3)+1

This is done for all 4 bases. The percentage composition of each base is also determined. Eight values are therefore determined, four position values and four composition values. These are then converted to probabilities (p) of coding using a look-up table provided as the data file for the program. The values in this look-up table have been determined experimentally using known coding and noncoding sequences.

Each of the probabilities is multiplied by a weight (w) value (again from the look-up table) for the respective base. The weight value reflects the percentage of the time that each parameter alone successfully predicted coding or noncoding function for the sequences of known function.

The TESTCODE statistic is then:

      p1w1 + p2w2 + p3w3 + p4w4 + p5w5 + p6w6 + p7w7 + p8w8

A result of less than 0.74 is probably a non-coding region.
A result equal or greater than 0.95 is probably a coding region.
Anything in between these two values is uncertain.

## Usage

Here is a sample session with tcode

    % tcode
    Identify protein-coding regions using Fickett TESTCODE statistic
    Input nucleotide sequence(s): tembl:x65921
    Length of sliding window [200]:
    Output report [x65921.tcode]:

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

Example 2

Produce a graphical plot

    % tcode -plot -graph cps
    Identify protein-coding regions using Fickett TESTCODE statistic
    Input nucleotide sequence(s): tembl:x65921
    Length of sliding window [200]:

    Created tcode.ps

Go to the output files for this example

## Command line arguments

       Standard (Mandatory) qualifiers (* if not always prompted):
      [-sequence]          seqall     Nucleotide sequence(s) filename and optional
                                      format, or reference (input USA)
       -window             integer    [200] This is the number of nucleotide bases
                                      over which the TESTCODE statistic will be
                                      performed each time. The window will then
                                      slide along the sequence, covering the same
                                      number of bases each time. (Integer 200 or
                                      more)
    *  -outfile            report     [*.tcode] Output report file name
    *  -graph              xygraph    [$EMBOSS_GRAPHICS value, or x11] Graph type
                                      (ps, hpgl, hp7470, hp7580, meta, cps, x11,
                                      tekt, tek, none, data, xterm, png, gif)

       Additional (Optional) qualifiers: (none)
       Advanced (Unprompted) qualifiers:
       -datafile           datafile   [Etcode.dat] The default data file is
                                      Etcode.dat and contains coding probabilities
                                      for each base. The probabilities are for
                                      both positional and compositional
                                      information.
       -step               integer    [3] The selected window will, by default,
                                      slide along the nucleotide sequence by three
                                      bases at a time, retaining the frame
                                      (although the algorithm is not frame
                                      sensitive). This may be altered to increase
                                      or decrease the increment of the slide.
                                      (Integer 1 or more)
       -plot               toggle     [N] On selection a graph of the sequence (X
                                      axis) plotted against the coding score (Y
                                      axis) will be displayed. Sequence above the
                                      green line is coding, that below the red
                                      line is non-coding.

       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

       "-outfile" associated qualifiers
       -rformat            string     Report format
       -rname              string     Base file name
       -rextension         string     File name extension
       -rdirectory         string     Output directory
       -raccshow           boolean    Show accession number in the report
       -rdesshow           boolean    Show description in the report
       -rscoreshow         boolean    Show the score in the report
       -rstrandshow        boolean    Show the nucleotide strand in the report
       -rusashow           boolean    Show the full USA in the report
       -rmaxall            integer    Maximum total hits to report
       -rmaxseq            integer    Maximum hits to report for one sequence

       "-graph" associated qualifiers
       -gprompt            boolean    Graph prompting
       -gdesc              string     Graph description
       -gtitle             string     Graph title
       -gsubtitle          string     Graph subtitle
       -gxtitle            string     Graph x axis title
       -gytitle            string     Graph y axis title
       -goutfile           string     Output file for non interactive displays
       -gdirectory         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
-window | This is the number of nucleotide bases over which the TESTCODE statistic will be performed each time. The window will then slide along the sequence, covering the same number of bases each time. | Integer 200 or more | 200
-outfile | Output report file name | Report output file | <*>.tcode
-graph | Graph type | EMBOSS has a list of known devices, including ps, hpgl, hp7470, hp7580, meta, cps, x11, tekt, tek, none, data, xterm, png, gif | EMBOSS_GRAPHICS value, or x11
Additional (Optional) qualifiers | Allowed values | Default
(none)
Advanced (Unprompted) qualifiers | Allowed values | Default
-datafile | The default data file is Etcode.dat and contains coding probabilities for each base. The probabilities are for both positional and compositional information. | Data file | Etcode.dat
-step | The selected window will, by default, slide along the nucleotide sequence by three bases at a time, retaining the frame (although the algorithm is not frame sensitive). This may be altered to increase or decrease the increment of the slide. | Integer 1 or more | 3
-plot | On selection a graph of the sequence (X axis) plotted against the coding score (Y axis) will be displayed. Sequence above the green line is coding, that below the red line is non-coding. | Toggle value Yes/No | No

## Input file format

tcode reads any normal sequence USAs.

The program will ignore ambiguity codes in the nucleic acid sequence and just accept the four common bases. This is a function of the algorithm, and the data tables.

### Input files for usage example

'tembl:x65921' is a sequence entry in the example nucleic acid database 'tembl'

### Database entry: tembl:x65921

    ID   X65921; SV 1; linear; genomic DNA; STD; HUM; 2016 BP.
    XX
    AC   X65921; S45242;
    XX
    DT   13-MAY-1992 (Rel. 31, Created)
    DT   14-NOV-2006 (Rel. 89, Last updated, Version 7)
    XX
    DE   H.sapiens fau 1 gene
    XX
    KW   fau 1 gene.
    XX
    OS   Homo sapiens (human)
    OC   Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia;
    OC   Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae;
    OC   Homo.
    XX
    RN   [1]
    RP   1-2016
    RA   Kas K.;
    RT   ;
    RL   Submitted (29-APR-1992) to the EMBL/GenBank/DDBJ databases.
    RL   K. Kas, University of Antwerp, Dept of Biochemistry T3.22,
    RL   Universiteitsplein 1, 2610 Wilrijk, BELGIUM
    XX
    RN   [2]
    RP   1-2016
    RX   DOI; 10.1016/0006-291X(92)91286-Y.
    RX   PUBMED; 1326960.
    RA   Kas K., Michiels L., Merregaert J.;
    RT   "Genomic structure and expression of the human fau gene: encoding the
    RT   ribosomal protein S30 fused to a ubiquitin-like protein.";
    RL   Biochem. Biophys. Res. Commun. 187(2):927-933(1992).
    XX
    DR   GDB; 191789.
    DR   GDB; 191790.
    DR   GDB; 354872.
    DR   GDB; 4590236.
    XX
    FH   Key             Location/Qualifiers
    FH
    FT   source          1..2016
    FT                   /organism="Homo sapiens"
    FT                   /mol_type="genomic DNA"
    FT                   /clone_lib="CML cosmid"
    FT                   /clone="15.1"
    FT                   /db_xref="taxon:9606"
    FT   mRNA            join(408..504,774..856,951..1095,1557..1612,1787..>1912)
    FT                   /gene="fau 1"
    FT   exon            408..504
    FT                   /number=1

      [Part of this file has been deleted for brevity]

    FT                   RAKRRMQYNRRFVNVVPTFGKKKGPNANS"
    FT   intron          857..950
    FT                   /number=2
    FT   exon            951..1095
    FT                   /number=3
    FT   intron          1096..1556
    FT                   /number=3
    FT   exon            1557..1612
    FT                   /number=4
    FT   intron          1613..1786
    FT                   /number=4
    FT   exon            1787..>1912
    FT                   /number=5
    FT   polyA_signal    1938..1943
    XX
    SQ   Sequence 2016 BP; 421 A; 562 C; 538 G; 495 T; 0 other;
         ctaccatttt ccctctcgat tctatatgta cactcgggac aagttctcct gatcgaaaac        60
         ggcaaaacta aggccccaag taggaatgcc ttagttttcg gggttaacaa tgattaacac       120
         tgagcctcac acccacgcga tgccctcagc tcctcgctca gcgctctcac caacagccgt       180
         agcccgcagc cccgctggac accggttctc catccccgca gcgtagcccg gaacatggta       240
         gctgccatct ttacctgcta cgccagcctt ctgtgcgcgc aactgtctgg tcccgccccg       300
         tcctgcgcga gctgctgccc aggcaggttc gccggtgcga gcgtaaaggg gcggagctag       360
         gactgccttg ggcggtacaa atagcaggga accgcgcggt cgctcagcag tgacgtgaca       420
         cgcagcccac ggtctgtact gacgcgccct cgcttcttcc tctttctcga ctccatcttc       480
         gcggtagctg ggaccgccgt tcaggtaaga atggggcctt ggctggatcc gaagggcttg       540
         tagcaggttg gctgcggggt cagaaggcgc ggggggaacc gaagaacggg gcctgctccg       600
         tggccctgct ccagtcccta tccgaactcc ttgggaggca ctggccttcc gcacgtgagc       660
         cgccgcgacc accatcccgt cgcgatcgtt tctggaccgc tttccactcc caaatctcct       720
         ttatcccaga gcatttcttg gcttctctta caagccgtct tttctttact cagtcgccaa       780
         tatgcagctc tttgtccgcg cccaggagct acacaccttc gaggtgaccg gccaggaaac       840
         ggtcgcccag atcaaggtaa ggctgcttgg tgcgccctgg gttccatttt cttgtgctct       900
         tcactctcgc ggcccgaggg aacgcttacg agccttatct ttccctgtag gctcatgtag       960
         cctcactgga gggcattgcc ccggaagatc aagtcgtgct cctggcaggc gcgcccctgg      1020
         aggatgaggc cactctgggc cagtgcgggg tggaggccct gactaccctg gaagtagcag      1080
         gccgcatgct tggaggtgag tgagagagga atgttctttg aagtaccggt aagcgtctag      1140
         tgagtgtggg gtgcatagtc ctgacagctg agtgtcacac ctatggtaat agagtacttc      1200
         tcactgtctt cagttcagag tgattcttcc tgtttacatc cctcatgttg aacacagacg      1260
         tccatgggag actgagccag agtgtagttg tatttcagtc acatcacgag atcctagtct      1320
         ggttatcagc ttccacacta aaaattaggt cagaccaggc cccaaagtgc tctataaatt      1380
         agaagctgga agatcctgaa atgaaactta agatttcaag gtcaaatatc tgcaactttg      1440
         ttctcattac ctattgggcg cagcttctct ttaaaggctt gaattgagaa aagaggggtt      1500
         ctgctgggtg gcaccttctt gctcttacct gctggtgcct tcctttccca ctacaggtaa      1560
         agtccatggt tccctggccc gtgctggaaa agtgagaggt cagactccta aggtgagtga      1620
         gagtattagt ggtcatggtg ttaggacttt ttttcctttc acagctaaac caagtccctg      1680
         ggctcttact cggtttgcct tctccctccc tggagatgag cctgagggaa gggatgctag      1740
         gtgtggaaga caggaaccag ggcctgatta accttccctt ctccaggtgg ccaaacagga      1800
         gaagaagaag aagaagacag gtcgggctaa gcggcggatg cagtacaacc ggcgctttgt      1860
         caacgttgtg cccacctttg gcaagaagaa gggccccaat gccaactctt aagtcttttg      1920
         taattctggc tttctctaat aaaaaagcca cttagttcag tcatcgcatt gtttcatctt      1980
         tacttgcaag gcctcaggga gaggtgtgct tctcgg                                2016
    //

## Output file format

The output is a standard EMBOSS report file.

The results can be output in one of several styles by using the command-line qualifier -rformat xxx, where 'xxx' is replaced by the name of the required format. The available format names are: embl, genbank, gff, pir, swiss, trace, listfile, dbmotif, diffseq, excel, feattable, motif, regions, seqtable, simple, srs, table, tagseq

See: http://emboss.sf.net/docs/themes/ReportFormats.html for further information on report formats.

tcode outputs a report format file. The default format is 'table'.

The resulting report file will be given a name relating to the analysed sequence together with the .tcode suffix by default. Should there be no sequence description, the default reverts to outfile.tcode.

tcode optionally outputs a graph to the specified graphics device.

The graphical display is output with the default file name tcode.1. and then the name of the selected graphical display (e.g. png; ps).

The graph indicates the threshold for probably being coding with a green horizontal line and the threshold for probably not being coding with a red horizontal line.

### Output files for usage example

### File: x65921.tcode

    ########################################
    # Program: tcode
    # Rundate: Tue 15 Jul 2008 12:00:00
    # Commandline: tcode
    #    -sequence tembl:x65921
    # Report_format: table
    # Report_file: x65921.tcode
    ########################################

    #=======================================
    #
    # Sequence: X65921     from: 1   to: 2016
    # HitCount: 606
    #
    # Fickett TESTCODE statistic
    #
    #=======================================

      Start     End  Strand   Score Estimation
          1     200       +   0.673 Non-coding
          4     203       +   0.681 Non-coding
          7     206       +   0.649 Non-coding
         10     209       +   0.649 Non-coding
         13     212       +   0.649 Non-coding
         16     215       +   0.657 Non-coding
         19     218       +   0.687 Non-coding
         22     221       +   0.767 No opinion
         25     224       +   0.800 No opinion
         28     227       +   0.782 No opinion
         31     230       +   0.779 No opinion
         34     233       +   0.839 No opinion
         37     236       +   0.951 Coding
         40     239       +   0.916 No opinion
         43     242       +   0.900 No opinion
         46     245       +   0.839 No opinion
         49     248       +   0.835 No opinion
         52     251       +   0.926 No opinion
         55     254       +   0.887 No opinion
         58     257       +   0.927 No opinion
         61     260       +   0.873 No opinion
         64     263       +   0.873 No opinion
         67     266       +   0.927 No opinion
         70     269       +   1.033 Coding
         73     272       +   1.044 Coding
         76     275       +   1.012 Coding
         79     278       +   1.105 Coding
         82     281       +   1.022 Coding
         85     284       +   1.098 Coding
         88     287       +   0.976 Coding
         91     290       +   0.962 Coding

      [Part of this file has been deleted for brevity]

       1690    1889       +   0.907 No opinion
       1693    1892       +   1.005 Coding
       1696    1895       +   0.985 Coding
       1699    1898       +   0.907 No opinion
       1702    1901       +   0.985 Coding
       1705    1904       +   0.985 Coding
       1708    1907       +   0.988 Coding
       1711    1910       +   0.981 Coding
       1714    1913       +   1.001 Coding
       1717    1916       +   1.001 Coding
       1720    1919       +   1.004 Coding
       1723    1922       +   0.975 Coding
       1726    1925       +   0.975 Coding
       1729    1928       +   0.927 No opinion
       1732    1931       +   0.939 No opinion
       1735    1934       +   0.930 No opinion
       1738    1937       +   0.860 No opinion
       1741    1940       +   0.876 No opinion
       1744    1943       +   0.876 No opinion
       1747    1946       +   0.836 No opinion
       1750    1949       +   0.883 No opinion
       1753    1952       +   0.934 No opinion
       1756    1955       +   0.965 Coding
       1759    1958       +   1.012 Coding
       1762    1961       +   1.025 Coding
       1765    1964       +   0.940 No opinion
       1768    1967       +   0.978 Coding
       1771    1970       +   0.978 Coding
       1774    1973       +   1.012 Coding
       1777    1976       +   1.023 Coding
       1780    1979       +   1.023 Coding
       1783    1982       +   1.023 Coding
       1786    1985       +   1.012 Coding
       1789    1988       +   0.972 Coding
       1792    1991       +   0.943 No opinion
       1795    1994       +   1.031 Coding
       1798    1997       +   1.031 Coding
       1801    2000       +   0.926 No opinion
       1804    2003       +   0.845 No opinion
       1807    2006       +   0.804 No opinion
       1810    2009       +   0.765 No opinion
       1813    2012       +   0.765 No opinion
       1816    2015       +   0.765 No opinion

    #---------------------------------------
    #---------------------------------------

    #---------------------------------------
    # Total_sequences: 1
    # Total_hitcount: 606
    #---------------------------------------

### Output files for usage example 2

### Graphics File: tcode.ps

## Data files

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 default data file (look-up table) is Etcode.dat which contains the data from the original paper (1)

    # Fickett TESTCODE data
    # Nuc. Acids Res. 10(17) 5303-5318
    #
    # Position parameter values (last value must be 0.0)
    1.9
    1.8
    1.7
    1.6
    1.5
    1.4
    1.3
    1.2
    1.1
    0.0
    #
    #
    # Content parameter values (last value must be 0.0)
    0.33
    0.31
    0.29
    0.27
    0.25
    0.23
    0.21
    0.17
    0.00
    #
    #
    # Position probabilities for A,C,G,T respectively
    0.94 0.80 0.90 0.97
    0.68 0.70 0.88 0.97
    0.84 0.70 0.74 0.91
    0.93 0.81 0.64 0.68
    0.58 0.66 0.53 0.69
    0.68 0.48 0.48 0.44
    0.45 0.51 0.27 0.54
    0.34 0.33 0.16 0.20
    0.20 0.30 0.08 0.09
    0.22 0.23 0.08 0.09
    #
    #
    # Content probabilities for A,C,G,T respectively
    0.28 0.82 0.40 0.28
    0.49 0.64 0.54 0.24
    0.44 0.51 0.47 0.39
    0.55 0.64 0.64 0.40
    0.62 0.59 0.64 0.55
    0.49 0.59 0.73 0.75
    0.67 0.43 0.41 0.56
    0.65 0.44 0.41 0.69
    0.81 0.39 0.33 0.51
    0.21 0.31 0.29 0.58
    #
    #
    # Weights for position
    0.26
    0.18
    0.31
    0.33
    #
    #
    # Weights for content
    0.11
    0.12
    0.15
    0.14

This file is retrievable using EMBOSSDATA.

Window size is set by default to 200. The algorithm requires sufficient sequence to perform the statistic on. The original paper suggests a minimum window size of 200.

Window stepping increment is set by default to 3. This will ensure the resulting information remains in frame.

### Alternative Data Files

There are no alternative data files currently in the EMBOSS Data directory, but alternative values may be user defined.

## Notes

The TESTCODE statistic reflects the fact that codons are used with unequal frequency and that oligonucleotides and nucleotides tend to be repeated with a periodicity of three. The original paper reports that the test had been thoroughly proven on 400,000 bases of sequence data: it misclassifies 5% of the regions tested and gives an answer of "No Opinion" one fifth of the time.

In the GCG package, the current (version 10.3) TESTCODE application's apparent interpretation of the algorithm is: MAX(A1,A2,A3) / MIN(A1,A2,A3) The EMBOSS tcode program uses the correct Fickett algorithm equation: MAX(A1,A2,A3) / MIN(A1,A2,A3) + 1 thus any plot using the GCG TESTCODE aplication will be slightly higher than the tcode equivalent.

## References

1. Fickett, J.W. (1982) Nucleic Acids Research 10(17) pp.5303-5318 "Recognition of protein coding regions in DNA sequences"

## Warnings

The program will ignore ambiguity codes in the nucleic acid sequence and just accept the four common bases. This is a function of the algorithm, and the data tables.

## Diagnostic Error Messages

Standard error messages are given for incorrect sequence input.

## Exit status

It always exits with status 0.

## See also

Program name | Description
--- | ---
getorf | Finds and extracts open reading frames (ORFs)
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
wobble | Plot third base position variability in a nucleotide sequence

### See Elsewhere

TESTCODE - GCG package, Accelrys Inc. Uses a different interpretation of the same algorithm. Source code unavailable.

SPIN - "Uneven positional base preferences" Staden software. Free to academics, versions for both X and Windows platforms.

## Author(s)

Alan Bleasby (ajb © ebi.ac.uk)
European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK

## History

Date of original completion: 2nd March 2003

## Target users

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

## Comments

None