| dan

## Wiki

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Please help by correcting and extending the Wiki pages.

## Function

Calculate nucleic acid melting temperature

## Description

Dan calculates the melting temperature (Tm) and the percentage of G+C nucleotides for windows over a nucleic acid sequence, optionally plotting them. If a plot is not being produced, dan reports the sequence of each oligomer window, its melting temperature under the specified conditions and its percentage GC content. The change in enthalpy (H), entropy (S) and Gibbs free energy (S) for dissociation of the oligomers may (optionally) be reported to file (but not plotted).

## Algorithm

The values of melting point and other thermodynamic properties of the sequence, namely change in enthalpy (H), entropy (S) and Gibbs free energy (S) on dissociation, are calculated for a sequence region (or "window") of a user-specified size (see "-windowsize" option). The window is incrementally moved along the sequence with the properties being calculated at each new position. The user must provide the salt and DNA concentration. Optionally, the percent formamide, percent of mismatches allowed and product length may be specified.

For the melting temperature profile, free energy values calculated from nearest neighbor thermodynamics are used (Breslauer et al. Proc. Natl. Acad. Sci. USA 83, 3746-3750, Baldino et al. Methods in Enzymol. 168, 761-777, Allawi and SantaLucia (1997), Biochemistry 36:10581-10594).

## Usage

Here is a sample session with dan

    % dan
    Calculate nucleic acid melting temperature
    Input nucleotide sequence(s): tembl:x13776
    Enter window size [20]:
    Enter shift increment [1]:
    Enter DNA concentration (nM) [50.]:
    Enter salt concentration (mM) [50.]:
    Output report [x13776.dan]:

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

Example 2

An example of producing a plot of Tm:

    % dan -plot -graph cps
    Calculate nucleic acid melting temperature
    Input nucleotide sequence(s): tembl:x13776
    Enter window size [20]:
    Enter shift increment [1]:
    Enter DNA concentration (nM) [50.]:
    Enter salt concentration (mM) [50.]:
    Enter minimum temperature [55.]:

    Created dan.ps

Go to the output files for this example

## Command line arguments

    Calculate nucleic acid melting temperature
    Version: EMBOSS:6.6.0.0

       Standard (Mandatory) qualifiers (* if not always prompted):
      [-sequence]          seqall     Nucleotide sequence(s) filename and optional
                                      format, or reference (input USA)
       -windowsize         integer    [20] The values of melting point and other
                                      thermodynamic properties of the sequence are
                                      determined by taking a short length of
                                      sequence known as a window and determining
                                      the properties of the sequence in that
                                      window. The window is incrementally moved
                                      along the sequence with the properties being
                                      calculated at each new position. (Integer
                                      from 1 to 100)
       -shiftincrement     integer    [1] This is the amount by which the window
                                      is moved at each increment in order to find
                                      the melting point and other properties along
                                      the sequence. (Integer 1 or more)
       -dnaconc            float      [50.] Enter DNA concentration (nM) (Number
                                      from 1.000 to 100000.000)
       -saltconc           float      [50.] Enter salt concentration (mM) (Number
                                      from 1.000 to 1000.000)
    *  -mintemp            float      [55.] Enter a minimum value for the
                                      temperature scale (y-axis) of the plot.
                                      (Number from 0.000 to 150.000)
    *  -graph              xygraph    [$EMBOSS_GRAPHICS value, or x11] Graph type
                                      (ps, hpgl, hp7470, hp7580, meta, cps, x11,
                                      tek, tekt, none, data, xterm, png, gif, pdf,
                                      svg)
    *  -outfile            report     [*.dan] If a plot is not being produced then
                                      data on the melting point etc. in each
                                      window along the sequence is output to the
                                      file. (default -rformat seqtable)

       Additional (Optional) qualifiers (* if not always prompted):
       -product            toggle     This prompts for percent formamide, percent
                                      of mismatches allowed and product length.
    *  -formamide          float      [0.] This specifies the percent formamide to
                                      be used in calculations (it is ignored
                                      unless -product is used). (Number from 0.000
                                      to 100.000)
    *  -mismatch           float      [0.] This specifies the percent mismatch to
                                      be used in calculations (it is ignored
                                      unless -product is used). (Number from 0.000
                                      to 100.000)
    *  -prodlen            integer    [Window size (20)] This specifies the
                                      product length to be used in calculations
                                      (it is ignored unless -product is used).
                                      (Any integer value)
       -thermo             toggle     Output the DeltaG, DeltaH and DeltaS values
                                      of the sequence windows to the output data
                                      file.
    *  -temperature        float      [25.] If -thermo has been specified then
                                      this specifies the temperature at which to
                                      calculate the DeltaG, DeltaH and DeltaS
                                      values. (Number from 0.000 to 100.000)

       Advanced (Unprompted) qualifiers:
       -rna                boolean    This specifies that the sequence is an RNA
                                      sequence and not a DNA sequence.
       -plot               toggle     If this is not specified then the file of
                                      output data is produced, else a plot of the
                                      melting point along the sequence is
                                      produced.

       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
       -scircular1         boolean    Sequence is circular
       -squick1            boolean    Read id and sequence only
       -sformat1           string     Input sequence format
       -iquery1            string     Input query fields or ID list
       -ioffset1           integer    Input start position offset
       -sdbname1           string     Database name
       -sid1               string     Entryname
       -ufo1               string     UFO features
       -fformat1           string     Features format
       -fopenfile1         string     Features file name

       "-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

       "-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

       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 and exit. 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
       -version            boolean    Report version number and exit

Qualifier | Type | Description | Allowed values | Default
--- | --- | --- | --- | ---
Standard (Mandatory) qualifiers
[-sequence] (Parameter 1) | seqall | Nucleotide sequence(s) filename and optional format, or reference (input USA) | Readable sequence(s) | Required
-windowsize | integer | The values of melting point and other thermodynamic properties of the sequence are determined by taking a short length of sequence known as a window and determining the properties of the sequence in that window. The window is incrementally moved along the sequence with the properties being calculated at each new position. | Integer from 1 to 100 | 20
-shiftincrement | integer | This is the amount by which the window is moved at each increment in order to find the melting point and other properties along the sequence. | Integer 1 or more | 1
-dnaconc | float | Enter DNA concentration (nM) | Number from 1.000 to 100000.000 | 50.
-saltconc | float | Enter salt concentration (mM) | Number from 1.000 to 1000.000 | 50.
-mintemp | float | Enter a minimum value for the temperature scale (y-axis) of the plot. | Number from 0.000 to 150.000 | 55.
-graph | xygraph | Graph type | EMBOSS has a list of known devices, including ps, hpgl, hp7470, hp7580, meta, cps, x11, tek, tekt, none, data, xterm, png, gif, pdf, svg | EMBOSS_GRAPHICS value, or x11
-outfile | report | If a plot is not being produced then data on the melting point etc. in each window along the sequence is output to the file. | (default -rformat seqtable) | <*>.dan
Additional (Optional) qualifiers
-product | toggle | This prompts for percent formamide, percent of mismatches allowed and product length. | Toggle value Yes/No | No
-formamide | float | This specifies the percent formamide to be used in calculations (it is ignored unless -product is used). | Number from 0.000 to 100.000 | 0.
-mismatch | float | This specifies the percent mismatch to be used in calculations (it is ignored unless -product is used). | Number from 0.000 to 100.000 | 0.
-prodlen | integer | This specifies the product length to be used in calculations (it is ignored unless -product is used). | Any integer value | Window size (20)
-thermo | toggle | Output the DeltaG, DeltaH and DeltaS values of the sequence windows to the output data file. | Toggle value Yes/No | No
-temperature | float | If -thermo has been specified then this specifies the temperature at which to calculate the DeltaG, DeltaH and DeltaS values. | Number from 0.000 to 100.000 | 25.
Advanced (Unprompted) qualifiers
-rna | boolean | This specifies that the sequence is an RNA sequence and not a DNA sequence. | Boolean value Yes/No | No
-plot | toggle | If this is not specified then the file of output data is produced, else a plot of the melting point along the sequence is produced. | Toggle value Yes/No | No
Associated qualifiers
"-sequence" associated seqall qualifiers
-sbegin1 -sbegin_sequence | integer | Start of each sequence to be used | Any integer value | 0
-send1 -send_sequence | integer | End of each sequence to be used | Any integer value | 0
-sreverse1 -sreverse_sequence | boolean | Reverse (if DNA) | Boolean value Yes/No | N
-sask1 -sask_sequence | boolean | Ask for begin/end/reverse | Boolean value Yes/No | N
-snucleotide1 -snucleotide_sequence | boolean | Sequence is nucleotide | Boolean value Yes/No | N
-sprotein1 -sprotein_sequence | boolean | Sequence is protein | Boolean value Yes/No | N
-slower1 -slower_sequence | boolean | Make lower case | Boolean value Yes/No | N
-supper1 -supper_sequence | boolean | Make upper case | Boolean value Yes/No | N
-scircular1 -scircular_sequence | boolean | Sequence is circular | Boolean value Yes/No | N
-squick1 -squick_sequence | boolean | Read id and sequence only | Boolean value Yes/No | N
-sformat1 -sformat_sequence | string | Input sequence format | Any string |
-iquery1 -iquery_sequence | string | Input query fields or ID list | Any string |
-ioffset1 -ioffset_sequence | integer | Input start position offset | Any integer value | 0
-sdbname1 -sdbname_sequence | string | Database name | Any string |
-sid1 -sid_sequence | string | Entryname | Any string |
-ufo1 -ufo_sequence | string | UFO features | Any string |
-fformat1 -fformat_sequence | string | Features format | Any string |
-fopenfile1 -fopenfile_sequence | string | Features file name | Any string |
"-graph" associated xygraph qualifiers
-gprompt | boolean | Graph prompting | Boolean value Yes/No | N
-gdesc | string | Graph description | Any string | DNA melting plot
-gtitle | string | Graph title | Any string |
-gsubtitle | string | Graph subtitle | Any string |
-gxtitle | string | Graph x axis title | Any string |
-gytitle | string | Graph y axis title | Any string |
-goutfile | string | Output file for non interactive displays | Any string |
-gdirectory | string | Output directory | Any string |
"-outfile" associated report qualifiers
-rformat | string | Report format | Any string | seqtable
-rname | string | Base file name | Any string |
-rextension | string | File name extension | Any string |
-rdirectory | string | Output directory | Any string |
-raccshow | boolean | Show accession number in the report | Boolean value Yes/No | N
-rdesshow | boolean | Show description in the report | Boolean value Yes/No | N
-rscoreshow | boolean | Show the score in the report | Boolean value Yes/No | Y
-rstrandshow | boolean | Show the nucleotide strand in the report | Boolean value Yes/No | Y
-rusashow | boolean | Show the full USA in the report | Boolean value Yes/No | N
-rmaxall | integer | Maximum total hits to report | Any integer value | 0
-rmaxseq | integer | Maximum hits to report for one sequence | Any integer value | 0
General qualifiers
-auto | boolean | Turn off prompts | Boolean value Yes/No | N
-stdout | boolean | Write first file to standard output | Boolean value Yes/No | N
-filter | boolean | Read first file from standard input, write first file to standard output | Boolean value Yes/No | N
-options | boolean | Prompt for standard and additional values | Boolean value Yes/No | N
-debug | boolean | Write debug output to program.dbg | Boolean value Yes/No | N
-verbose | boolean | Report some/full command line options | Boolean value Yes/No | Y
-help | boolean | Report command line options and exit. More information on associated and general qualifiers can be found with -help -verbose | Boolean value Yes/No | N
-warning | boolean | Report warnings | Boolean value Yes/No | Y
-error | boolean | Report errors | Boolean value Yes/No | Y
-fatal | boolean | Report fatal errors | Boolean value Yes/No | Y
-die | boolean | Report dying program messages | Boolean value Yes/No | Y
-version | boolean | Report version number and exit | Boolean value Yes/No | N

## Input file format

dan reads one or more nucleotide sequences.

The input is a standard EMBOSS sequence query (also known as a 'USA').

Major sequence database sources defined as standard in EMBOSS installations include srs:embl, srs:uniprot and ensembl

Data can also be read from sequence output in any supported format written by an EMBOSS or third-party application.

The input format can be specified by using the command-line qualifier `-sformat xxx`, where 'xxx' is replaced by the name of the required format. The available format names are: gff (gff3), gff2, embl (em), genbank (gb, refseq), ddbj, refseqp, pir (nbrf), swissprot (swiss, sw), dasgff and debug.

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

### Input files for usage example

'tembl:x13776' is a sequence entry in the example nucleic acid database 'tembl'

### Database entry: tembl:x13776

    ID   X13776; SV 1; linear; genomic DNA; STD; PRO; 2167 BP.
    XX
    AC   X13776; M43175;
    XX
    DT   19-APR-1989 (Rel. 19, Created)
    DT   14-NOV-2006 (Rel. 89, Last updated, Version 24)
    XX
    DE   Pseudomonas aeruginosa amiC and amiR gene for aliphatic amidase regulation
    XX
    KW   aliphatic amidase regulator; amiC gene; amiR gene.
    XX
    OS   Pseudomonas aeruginosa
    OC   Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales;
    OC   Pseudomonadaceae; Pseudomonas.
    XX
    RN   [1]
    RP   1167-2167
    RA   Rice P.M.;
    RT   ;
    RL   Submitted (16-DEC-1988) to the INSDC.
    RL   Rice P.M., EMBL, Postfach 10-2209, Meyerhofstrasse 1, 6900 Heidelberg, FRG.
    XX
    RN   [2]
    RP   1167-2167
    RX   DOI; 10.1016/0014-5793(89)80249-2.
    RX   PUBMED; 2495988.
    RA   Lowe N., Rice P.M., Drew R.E.;
    RT   "Nucleotide sequence of the aliphatic amidase regulator gene (amiR) of
    RT   Pseudomonas aeruginosa";
    RL   FEBS Lett. 246(1-2):39-43(1989).
    XX
    RN   [3]
    RP   1-1292
    RX   PUBMED; 1907262.
    RA   Wilson S., Drew R.;
    RT   "Cloning and DNA sequence of amiC, a new gene regulating expression of the
    RT   Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC
    RT   product";
    RL   J. Bacteriol. 173(16):4914-4921(1991).
    XX
    RN   [4]
    RP   1-2167
    RA   Rice P.M.;
    RT   ;
    RL   Submitted (04-SEP-1991) to the INSDC.
    RL   Rice P.M., EMBL, Postfach 10-2209, Meyerhofstrasse 1, 6900 Heidelberg, FRG.
    XX
    DR   GOA; Q51417.
    DR   InterPro; IPR003211; AmiSUreI_transpt.
    DR   UniProtKB/Swiss-Prot; Q51417; AMIS_PSEAE.

      [Part of this file has been deleted for brevity]

    FT                   /note="ClaI fragment deleted in pSW36,  constitutive
    FT                   phenotype"
    FT   misc_feature    1
    FT                   /note="last base of an XhoI site"
    FT   misc_feature    648..653
    FT                   /note="end of 658bp XhoI fragment, deletion in  pSW3 causes
    FT                   constitutive expression of amiE"
    FT   misc_difference 1281
    FT                   /replace="g"
    FT                   /note="conflict"
    FT                   /citation=[3]
    XX
    SQ   Sequence 2167 BP; 363 A; 712 C; 730 G; 362 T; 0 other;
         ggtaccgctg gccgagcatc tgctcgatca ccaccagccg ggcgacggga actgcacgat        60
         ctacctggcg agcctggagc acgagcgggt tcgcttcgta cggcgctgag cgacagtcac       120
         aggagaggaa acggatggga tcgcaccagg agcggccgct gatcggcctg ctgttctccg       180
         aaaccggcgt caccgccgat atcgagcgct cgcacgcgta tggcgcattg ctcgcggtcg       240
         agcaactgaa ccgcgagggc ggcgtcggcg gtcgcccgat cgaaacgctg tcccaggacc       300
         ccggcggcga cccggaccgc tatcggctgt gcgccgagga cttcattcgc aaccgggggg       360
         tacggttcct cgtgggctgc tacatgtcgc acacgcgcaa ggcggtgatg ccggtggtcg       420
         agcgcgccga cgcgctgctc tgctacccga ccccctacga gggcttcgag tattcgccga       480
         acatcgtcta cggcggtccg gcgccgaacc agaacagtgc gccgctggcg gcgtacctga       540
         ttcgccacta cggcgagcgg gtggtgttca tcggctcgga ctacatctat ccgcgggaaa       600
         gcaaccatgt gatgcgccac ctgtatcgcc agcacggcgg cacggtgctc gaggaaatct       660
         acattccgct gtatccctcc gacgacgact tgcagcgcgc cgtcgagcgc atctaccagg       720
         cgcgcgccga cgtggtcttc tccaccgtgg tgggcaccgg caccgccgag ctgtatcgcg       780
         ccatcgcccg tcgctacggc gacggcaggc ggccgccgat cgccagcctg accaccagcg       840
         aggcggaggt ggcgaagatg gagagtgacg tggcagaggg gcaggtggtg gtcgcgcctt       900
         acttctccag catcgatacg cccgccagcc gggccttcgt ccaggcctgc catggtttct       960
         tcccggagaa cgcgaccatc accgcctggg ccgaggcggc ctactggcag accttgttgc      1020
         tcggccgcgc cgcgcaggcc gcaggcaact ggcgggtgga agacgtgcag cggcacctgt      1080
         acgacatcga catcgacgcg ccacaggggc cggtccgggt ggagcgccag aacaaccaca      1140
         gccgcctgtc ttcgcgcatc gcggaaatcg atgcgcgcgg cgtgttccag gtccgctggc      1200
         agtcgcccga accgattcgc cccgaccctt atgtcgtcgt gcataacctc gacgactggt      1260
         ccgccagcat gggcggggga ccgctcccat gagcgccaac tcgctgctcg gcagcctgcg      1320
         cgagttgcag gtgctggtcc tcaacccgcc gggggaggtc agcgacgccc tggtcttgca      1380
         gctgatccgc atcggttgtt cggtgcgcca gtgctggccg ccgccggaag ccttcgacgt      1440
         gccggtggac gtggtcttca ccagcatttt ccagaatggc caccacgacg agatcgctgc      1500
         gctgctcgcc gccgggactc cgcgcactac cctggtggcg ctggtggagt acgaaagccc      1560
         cgcggtgctc tcgcagatca tcgagctgga gtgccacggc gtgatcaccc agccgctcga      1620
         tgcccaccgg gtgctgcctg tgctggtatc ggcgcggcgc atcagcgagg aaatggcgaa      1680
         gctgaagcag aagaccgagc agctccagga ccgcatcgcc ggccaggccc ggatcaacca      1740
         ggccaaggtg ttgctgatgc agcgccatgg ctgggacgag cgcgaggcgc accagcacct      1800
         gtcgcgggaa gcgatgaagc ggcgcgagcc gatcctgaag atcgctcagg agttgctggg      1860
         aaacgagccg tccgcctgag cgatccgggc cgaccagaac aataacaaga ggggtatcgt      1920
         catcatgctg ggactggttc tgctgtacgt tggcgcggtg ctgtttctca atgccgtctg      1980
         gttgctgggc aagatcagcg gtcgggaggt ggcggtgatc aacttcctgg tcggcgtgct      2040
         gagcgcctgc gtcgcgttct acctgatctt ttccgcagca gccgggcagg gctcgctgaa      2100
         ggccggagcg ctgaccctgc tattcgcttt tacctatctg tgggtggccg ccaaccagtt      2160
         cctcgag                                                                2167
    //

## Output file format

If a plot is not being produced, dan reports the sequence of each oligomer window, its melting temperature under the specified conditions and its GC content.

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, dasgff, debug, listfile, dbmotif, diffseq, draw, restrict, excel, feattable, motif, nametable, regions, seqtable, simple, srs, table, tagseq.

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

By default dan writes a 'seqtable' report file.

The output is to the specified graphics device.

The results can be output in one of several formats by using the command-line qualifier -graph xxx, where 'xxx' is replaced by the name of the required device. Support depends on the availability of third-party software packages.

The device names that output to a file are: ps (postscript), cps (colourps), png, gif, pdf, svg, hpgl, hp7470, hp7580, das, data.

The other available device names are: meta, x11 (xwindows), tek (tek4107t), tekt (tektronix), xterm, text.

Output can be turned off by specifying none (null).

See: http://emboss.sf.net/docs/themes/GraphicsDevices.html for further information on supported devices.

If the `-plot` qualifier is used, graphical output is produced instead.

### Output files for usage example

### File: x13776.dan

    ########################################
    # Program: dan
    # Rundate: Mon 15 Jul 2013 12:00:00
    # Commandline: dan
    #    -sequence tembl:x13776
    # Report_format: seqtable
    # Report_file: x13776.dan
    ########################################

    #=======================================
    #
    # Sequence: X13776     from: 1   to: 2167
    # HitCount: 2148
    #=======================================

      Start     End  Strand     Tm     GC DeltaG DeltaH DeltaS TmProd Sequence
          1      20       +   64.9   70.0      .      .      .      . ggtaccgctggccgagcatc
          2      21       +   63.7   65.0      .      .      .      . gtaccgctggccgagcatct
          3      22       +   63.7   65.0      .      .      .      . taccgctggccgagcatctg
          4      23       +   66.9   70.0      .      .      .      . accgctggccgagcatctgc
          5      24       +   66.7   70.0      .      .      .      . ccgctggccgagcatctgct
          6      25       +   65.5   70.0      .      .      .      . cgctggccgagcatctgctc
          7      26       +   65.5   70.0      .      .      .      . gctggccgagcatctgctcg
          8      27       +   63.7   65.0      .      .      .      . ctggccgagcatctgctcga
          9      28       +   62.9   60.0      .      .      .      . tggccgagcatctgctcgat
         10      29       +   62.6   65.0      .      .      .      . ggccgagcatctgctcgatc
         11      30       +   61.7   60.0      .      .      .      . gccgagcatctgctcgatca
         12      31       +   60.2   60.0      .      .      .      . ccgagcatctgctcgatcac
         13      32       +   60.2   60.0      .      .      .      . cgagcatctgctcgatcacc
         14      33       +   59.0   55.0      .      .      .      . gagcatctgctcgatcacca
         15      34       +   59.2   55.0      .      .      .      . agcatctgctcgatcaccac
         16      35       +   60.4   60.0      .      .      .      . gcatctgctcgatcaccacc
         17      36       +   58.9   55.0      .      .      .      . catctgctcgatcaccacca
         18      37       +   58.6   55.0      .      .      .      . atctgctcgatcaccaccag
         19      38       +   61.3   60.0      .      .      .      . tctgctcgatcaccaccagc
         20      39       +   62.4   65.0      .      .      .      . ctgctcgatcaccaccagcc
         21      40       +   63.9   65.0      .      .      .      . tgctcgatcaccaccagccg
         22      41       +   64.9   70.0      .      .      .      . gctcgatcaccaccagccgg
         23      42       +   64.3   70.0      .      .      .      . ctcgatcaccaccagccggg
         24      43       +   66.1   70.0      .      .      .      . tcgatcaccaccagccgggc
         25      44       +   67.5   75.0      .      .      .      . cgatcaccaccagccgggcg
         26      45       +   66.1   70.0      .      .      .      . gatcaccaccagccgggcga
         27      46       +   66.3   70.0      .      .      .      . atcaccaccagccgggcgac
         28      47       +   68.6   75.0      .      .      .      . tcaccaccagccgggcgacg
         29      48       +   69.8   80.0      .      .      .      . caccaccagccgggcgacgg
         30      49       +   70.7   80.0      .      .      .      . accaccagccgggcgacggg
         31      50       +   70.5   80.0      .      .      .      . ccaccagccgggcgacggga
         32      51       +   68.6   75.0      .      .      .      . caccagccgggcgacgggaa
         33      52       +   68.6   75.0      .      .      .      . accagccgggcgacgggaac
         34      53       +   68.4   75.0      .      .      .      . ccagccgggcgacgggaact

      [Part of this file has been deleted for brevity]

       2101    2120       +   69.9   80.0      .      .      .      . ggccggagcgctgaccctgc
       2102    2121       +   68.7   75.0      .      .      .      . gccggagcgctgaccctgct
       2103    2122       +   65.5   70.0      .      .      .      . ccggagcgctgaccctgcta
       2104    2123       +   63.5   65.0      .      .      .      . cggagcgctgaccctgctat
       2105    2124       +   61.3   60.0      .      .      .      . ggagcgctgaccctgctatt
       2106    2125       +   60.1   60.0      .      .      .      . gagcgctgaccctgctattc
       2107    2126       +   61.7   60.0      .      .      .      . agcgctgaccctgctattcg
       2108    2127       +   63.4   65.0      .      .      .      . gcgctgaccctgctattcgc
       2109    2128       +   61.7   60.0      .      .      .      . cgctgaccctgctattcgct
       2110    2129       +   59.5   55.0      .      .      .      . gctgaccctgctattcgctt
       2111    2130       +   57.1   50.0      .      .      .      . ctgaccctgctattcgcttt
       2112    2131       +   56.4   45.0      .      .      .      . tgaccctgctattcgctttt
       2113    2132       +   54.7   45.0      .      .      .      . gaccctgctattcgctttta
       2114    2133       +   55.0   45.0      .      .      .      . accctgctattcgcttttac
       2115    2134       +   55.9   50.0      .      .      .      . ccctgctattcgcttttacc
       2116    2135       +   54.7   45.0      .      .      .      . cctgctattcgcttttacct
       2117    2136       +   52.0   40.0      .      .      .      . ctgctattcgcttttaccta
       2118    2137       +   51.2   35.0      .      .      .      . tgctattcgcttttacctat
       2119    2138       +   50.9   40.0      .      .      .      . gctattcgcttttacctatc
       2120    2139       +   49.0   35.0      .      .      .      . ctattcgcttttacctatct
       2121    2140       +   49.3   35.0      .      .      .      . tattcgcttttacctatctg
       2122    2141       +   51.1   35.0      .      .      .      . attcgcttttacctatctgt
       2123    2142       +   52.2   40.0      .      .      .      . ttcgcttttacctatctgtg
       2124    2143       +   54.0   45.0      .      .      .      . tcgcttttacctatctgtgg
       2125    2144       +   55.2   50.0      .      .      .      . cgcttttacctatctgtggg
       2126    2145       +   53.9   45.0      .      .      .      . gcttttacctatctgtgggt
       2127    2146       +   52.3   45.0      .      .      .      . cttttacctatctgtgggtg
       2128    2147       +   53.5   45.0      .      .      .      . ttttacctatctgtgggtgg
       2129    2148       +   56.0   50.0      .      .      .      . tttacctatctgtgggtggc
       2130    2149       +   57.8   55.0      .      .      .      . ttacctatctgtgggtggcc
       2131    2150       +   60.1   60.0      .      .      .      . tacctatctgtgggtggccg
       2132    2151       +   63.4   65.0      .      .      .      . acctatctgtgggtggccgc
       2133    2152       +   64.3   70.0      .      .      .      . cctatctgtgggtggccgcc
       2134    2153       +   63.4   65.0      .      .      .      . ctatctgtgggtggccgcca
       2135    2154       +   62.7   60.0      .      .      .      . tatctgtgggtggccgccaa
       2136    2155       +   64.5   65.0      .      .      .      . atctgtgggtggccgccaac
       2137    2156       +   66.5   70.0      .      .      .      . tctgtgggtggccgccaacc
       2138    2157       +   66.8   70.0      .      .      .      . ctgtgggtggccgccaacca
       2139    2158       +   66.8   70.0      .      .      .      . tgtgggtggccgccaaccag
       2140    2159       +   66.8   70.0      .      .      .      . gtgggtggccgccaaccagt
       2141    2160       +   65.9   65.0      .      .      .      . tgggtggccgccaaccagtt
       2142    2161       +   65.6   70.0      .      .      .      . gggtggccgccaaccagttc
       2143    2162       +   65.6   70.0      .      .      .      . ggtggccgccaaccagttcc
       2144    2163       +   64.4   65.0      .      .      .      . gtggccgccaaccagttcct
       2145    2164       +   64.1   65.0      .      .      .      . tggccgccaaccagttcctc
       2146    2165       +   65.4   70.0      .      .      .      . ggccgccaaccagttcctcg
       2147    2166       +   64.2   65.0      .      .      .      . gccgccaaccagttcctcga
       2148    2167       +   62.4   65.0      .      .      .      . ccgccaaccagttcctcgag

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

### Output files for usage example 2

### Graphics File: dan.ps

The header information contains details of the program, date and sequence

Subsequent lines contain columns of data for each window into the sequence as it is moved along, giving:

- The start postion of the window
- The end position of the window
- The melting temperature of the window
- The percentage C+G of the window
- The sequence of the window

If the qualifier '-product' is used to make the program prompt for percent formamide percent of mismatches allowed and product length, then the output includes the melting temperature of the specified product.

If the qualifier '-thermo' is gived then the DeltaG, DeltaH and DeltaS of the sequence in the window is also output.

## Data files

The EMBOSS data files "Edna.melt" and "Erna.melt" are used to read in the entropy/enthalpy/energy data for DNA and RNA respectively.

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

## Notes

The enthalpy of a reaction equates to the "heat" of the reaction so long as temperate and pressure are constant. Enthalpy depends upon the strength of the chemical bonds and non-bonding interactions involved. The entropy of a reaction reflects the level of disorder or randomness of the molecules involved. The higher the entropy, the greater the disorder. The free energy of a reaction reflects it's ability to do work. It helps one determine whether a reaction is feasible given a set of conditions and it's quilibrium.

## References

1. Breslauer, K.J., Frank, R., Blocker, H., and Marky, L.A. (1986). "Predicting DNA Duplex Stability from the Base Sequence." Proceedings of the National Academy of Sciences USA 83, 3746-3750.
2. Baldino, M., Jr. (1989). "High Resolution In Situ Hybridization Histochemistry." In Methods in Enzymology, (P.M. Conn, ed.), 168, 761-777, Academic Press, San Diego, California, USA.
3. Allawi H.T. and SantaLucia J. Jr. (1997), "Thermodynamics and NMR of Internal G*T Mismatches in DNA" Biochemistry 36, 10581-10594.

## Warnings

RNA sequences must be submited to this application with the '-rna' qualifier on the command line, otherwise the sequence will be assumed to be DNA.

## Diagnostic Error Messages

None.

## Exit status

0 if successful.

## Known bugs

None.

## See also

Program name | Description
--- | ---
banana | Plot bending and curvature data for B-DNA
btwisted | Calculate the twisting in a B-DNA sequence
chaos | Draw a chaos game representation plot for a nucleotide sequence
compseq | Calculate the composition of unique words in sequences
density | Draw a nucleic acid density plot
freak | Generate residue/base frequency table or plot
isochore | Plot isochores in DNA sequences
wordcount | Count and extract unique words in molecular sequence(s)

## Author(s)

This program was originally included in EGCG under the names "MELT" and "MELTPLOT", written by Rodrigo Lopez
European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK

Please report all bugs to the EMBOSS bug team (emboss-bug © emboss.open-bio.org) not to the original author.

This application was written by Alan Bleasby
European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK

Please report all bugs to the EMBOSS bug team (emboss-bug © emboss.open-bio.org) not to the original author.

## History

Written (1999) - Alan Bleasby

## Target users

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

## Comments

None