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tcode |
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.
This application can assist in determining the probability of a region of nucleic sequence encoding a functional protein.
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.
% 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
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 |
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.
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
//
|
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.
########################################
# 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
#---------------------------------------
|
![[tcode results]](tcode.2.tcode.gif)
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 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.
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.
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.
| 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 |
SPIN - "Uneven positional base preferences" Staden software. Free to academics, versions for both X and Windows platforms.