EMBOSS

| diffseq

## Function

Compare and report features of two similar sequences

## Description

diffseq reads two sequences which typically are very similar or almost identical. It finds regions of identity (exact matches) in the two sequences and reports on similarities and differences between the features of the two sequences within these regions. The output is a standard EMBOSS report file. The start and end positions of the regions of identity are reported. Any features that are shared and any differerences in features are reported. The original feature table of each sequence may also (optionally) be written to file.

## Algorithm

diffseq searches for identical matches between all sequence words from both sequences. Identical sequence regions are found by creating a hash table of subsequences of user-defined size (-wordsize option), which is 10 by default. It then reduces the matches to a minimum set of overlapping matches by sorting them in order of size (largest size first). For each such match it removes any smaller matches that overlap. The result is a set of the longest regions of identity between the two sequences that do not overlap with each other. The mismatched regions between these matches are reported.

## Usage

Here is a sample session with diffseq

    % diffseq tembl:x65923 tembl:ay411291
    Compare and report features of two similar sequences
    Word size [10]:
    Output report [x65923.diffseq]:
    Features output [X65923.diffgff]:
    Second features output [AY411291.diffgff]:

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

## Command line arguments

       Standard (Mandatory) qualifiers:
      [-asequence]         sequence   Sequence filename and optional format, or
                                      reference (input USA)
      [-bsequence]         sequence   Sequence filename and optional format, or
                                      reference (input USA)
       -wordsize           integer    [10] The similar regions between the two
                                      sequences are found by creating a hash table
                                      of 'wordsize'd subsequences. 10 is a
                                      reasonable default. Making this value larger
                                      (20?) may speed up the program slightly,
                                      but will mean that any two differences
                                      within 'wordsize' of each other will be
                                      grouped as a single region of difference.
                                      This value may be made smaller (4?) to
                                      improve the resolution of nearby
                                      differences, but the program will go much
                                      slower. (Integer 2 or more)
      [-outfile]           report     [*.diffseq] Output report file name
      [-aoutfeat]          featout    [$(asequence.name).diffgff] File for output
                                      of first sequence's features
      [-boutfeat]          featout    [$(bsequence.name).diffgff] File for output
                                      of second sequence's features

       Additional (Optional) qualifiers:
       -globaldifferences  boolean    [N] Normally this program will find regions
                                      of identity that are the length of the
                                      specified word-size or greater and will then
                                      report the regions of difference between
                                      these matching regions. This works well and
                                      is what most people want if they are working
                                      with long overlapping nucleic acid
                                      sequences. You are usually not interested in
                                      the non-overlapping ends of these
                                      sequences. If you have protein sequences or
                                      short RNA sequences however, you will be
                                      interested in differences at the very ends .
                                      It this option is set to be true then the
                                      differences at the ends will also be
                                      reported.

       Advanced (Unprompted) qualifiers: (none)
       Associated qualifiers:

       "-asequence" associated qualifiers
       -sbegin1            integer    Start of the sequence to be used
       -send1              integer    End of the 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

       "-bsequence" associated qualifiers
       -sbegin2            integer    Start of the sequence to be used
       -send2              integer    End of the sequence to be used
       -sreverse2          boolean    Reverse (if DNA)
       -sask2              boolean    Ask for begin/end/reverse
       -snucleotide2       boolean    Sequence is nucleotide
       -sprotein2          boolean    Sequence is protein
       -slower2            boolean    Make lower case
       -supper2            boolean    Make upper case
       -sformat2           string     Input sequence format
       -sdbname2           string     Database name
       -sid2               string     Entryname
       -ufo2               string     UFO features
       -fformat2           string     Features format
       -fopenfile2         string     Features file name

       "-outfile" associated qualifiers
       -rformat3           string     Report format
       -rname3             string     Base file name
       -rextension3        string     File name extension
       -rdirectory3        string     Output directory
       -raccshow3          boolean    Show accession number in the report
       -rdesshow3          boolean    Show description in the report
       -rscoreshow3        boolean    Show the score in the report
       -rstrandshow3       boolean    Show the nucleotide strand in the report
       -rusashow3          boolean    Show the full USA in the report
       -rmaxall3           integer    Maximum total hits to report
       -rmaxseq3           integer    Maximum hits to report for one sequence

       "-aoutfeat" associated qualifiers
       -offormat4          string     Output feature format
       -ofopenfile4        string     Features file name
       -ofextension4       string     File name extension
       -ofdirectory4       string     Output directory
       -ofname4            string     Base file name
       -ofsingle4          boolean    Separate file for each entry

       "-boutfeat" associated qualifiers
       -offormat5          string     Output feature format
       -ofopenfile5        string     Features file name
       -ofextension5       string     File name extension
       -ofdirectory5       string     Output directory
       -ofname5            string     Base file name
       -ofsingle5          boolean    Separate file for each entry

       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
--- | --- | ---
[-asequence] (Parameter 1) | Sequence filename and optional format, or reference (input USA) | Readable sequence | Required
[-bsequence] (Parameter 2) | Sequence filename and optional format, or reference (input USA) | Readable sequence | Required
-wordsize | The similar regions between the two sequences are found by creating a hash table of 'wordsize'd subsequences. 10 is a reasonable default. Making this value larger (20?) may speed up the program slightly, but will mean that any two differences within 'wordsize' of each other will be grouped as a single region of difference. This value may be made smaller (4?) to improve the resolution of nearby differences, but the program will go much slower. | Integer 2 or more | 10
[-outfile] (Parameter 3) | Output report file name | Report output file | <*>.diffseq
[-aoutfeat] (Parameter 4) | File for output of first sequence's features | Writeable feature table | $(asequence.name).diffgff
[-boutfeat] (Parameter 5) | File for output of second sequence's features | Writeable feature table | $(bsequence.name).diffgff
Additional (Optional) qualifiers | Allowed values | Default
-globaldifferences | Normally this program will find regions of identity that are the length of the specified word-size or greater and will then report the regions of difference between these matching regions. This works well and is what most people want if they are working with long overlapping nucleic acid sequences. You are usually not interested in the non-overlapping ends of these sequences. If you have protein sequences or short RNA sequences however, you will be interested in differences at the very ends . It this option is set to be true then the differences at the ends will also be reported. | Boolean value Yes/No | No
Advanced (Unprompted) qualifiers | Allowed values | Default
(none)

## Input file format

This program reads in two nucleic acid sequence USAs or two protein sequence USAs.

### Input files for usage example

'tembl:x65923' is a sequence entry in the example nucleic acid database 'tembl'

### Database entry: tembl:x65923

    ID   X65923; SV 1; linear; mRNA; STD; HUM; 518 BP.
    XX
    AC   X65923;
    XX
    DT   13-MAY-1992 (Rel. 31, Created)
    DT   18-APR-2005 (Rel. 83, Last updated, Version 11)
    XX
    DE   H.sapiens fau mRNA
    XX
    KW   fau 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   1-518
    RA   Michiels L.M.R.;
    RT   ;
    RL   Submitted (29-APR-1992) to the EMBL/GenBank/DDBJ databases.
    RL   L.M.R. Michiels, University of Antwerp, Dept of Biochemistry,
    RL   Universiteisplein 1, 2610 Wilrijk, BELGIUM
    XX
    RN   [2]
    RP   1-518
    RX   PUBMED; 8395683.
    RA   Michiels L., Van der Rauwelaert E., Van Hasselt F., Kas K., Merregaert J.;
    RT   " fau cDNA encodes a ubiquitin-like-S30 fusion protein and is expressed as
    RT   an antisense sequences in the Finkel-Biskis-Reilly murine sarcoma virus";
    RL   Oncogene 8(9):2537-2546(1993).
    XX
    DR   H-InvDB; HIT000322806.
    XX
    FH   Key             Location/Qualifiers
    FH
    FT   source          1..518
    FT                   /organism="Homo sapiens"
    FT                   /chromosome="11q"
    FT                   /map="13"
    FT                   /mol_type="mRNA"
    FT                   /clone_lib="cDNA"
    FT                   /clone="pUIA 631"
    FT                   /tissue_type="placenta"
    FT                   /db_xref="taxon:9606"
    FT   misc_feature    57..278
    FT                   /note="ubiquitin like part"
    FT   CDS             57..458
    FT                   /gene="fau"
    FT                   /db_xref="GDB:135476"
    FT                   /db_xref="GOA:P35544"
    FT                   /db_xref="GOA:P62861"
    FT                   /db_xref="HGNC:3597"
    FT                   /db_xref="UniProtKB/Swiss-Prot:P35544"
    FT                   /db_xref="UniProtKB/Swiss-Prot:P62861"
    FT                   /protein_id="CAA46716.1"
    FT                   /translation="MQLFVRAQELHTFEVTGQETVAQIKAHVASLEGIAPEDQVVLLAG
    FT                   APLEDEATLGQCGVEALTTLEVAGRMLGGKVHGSLARAGKVRGQTPKVAKQEKKKKKTG
    FT                   RAKRRMQYNRRFVNVVPTFGKKKGPNANS"
    FT   misc_feature    98..102
    FT                   /note="nucleolar localization signal"
    FT   misc_feature    279..458
    FT                   /note="S30 part"
    FT   polyA_signal    484..489
    FT   polyA_site      509
    XX
    SQ   Sequence 518 BP; 125 A; 139 C; 148 G; 106 T; 0 other;
         ttcctctttc tcgactccat cttcgcggta gctgggaccg ccgttcagtc gccaatatgc        60
         agctctttgt ccgcgcccag gagctacaca ccttcgaggt gaccggccag gaaacggtcg       120
         cccagatcaa ggctcatgta gcctcactgg agggcattgc cccggaagat caagtcgtgc       180
         tcctggcagg cgcgcccctg gaggatgagg ccactctggg ccagtgcggg gtggaggccc       240
         tgactaccct ggaagtagca ggccgcatgc ttggaggtaa agttcatggt tccctggccc       300
         gtgctggaaa agtgagaggt cagactccta aggtggccaa acaggagaag aagaagaaga       360
         agacaggtcg ggctaagcgg cggatgcagt acaaccggcg ctttgtcaac gttgtgccca       420
         cctttggcaa gaagaagggc cccaatgcca actcttaagt cttttgtaat tctggctttc       480
         tctaataaaa aagccactta gttcagtcaa aaaaaaaa                               518
    //

### Database entry: tembl:ay411291

    ID   AY411291; SV 1; linear; genomic DNA; GSS; HUM; 402 BP.
    XX
    AC   AY411291;
    XX
    DT   13-DEC-2003 (Rel. 78, Created)
    DT   17-DEC-2003 (Rel. 78, Last updated, Version 2)
    XX
    DE   Homo sapiens FAU gene, VIRTUAL TRANSCRIPT, partial sequence, genomic survey
    DE   sequence.
    XX
    KW   GSS.
    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   1-402
    RX   DOI; 10.1126/science.1088821.
    RX   PUBMED; 14671302.
    RA   Clark A.G., Glanowski S., Nielson R., Thomas P., Kejariwal A., Todd M.A.,
    RA   Tanenbaum D.M., Civello D.R., Lu F., Murphy B., Ferriera S., Wang G.,
    RA   Zheng X.H., White T.J., Sninsky J.J., Adams M.D., Cargill M.;
    RT   "Inferring nonneutral evolution from human-chimp-mouse orthologous gene
    RT   trios";
    RL   Science 302(5652):1960-1963(2003).
    XX
    RN   [2]
    RP   1-402
    RA   Clark A.G., Glanowski S., Nielson R., Thomas P., Kejariwal A., Todd M.A.,
    RA   Tanenbaum D.M., Civello D.R., Lu F., Murphy B., Ferriera S., Wang G.,
    RA   Zheng X.H., White T.J., Sninsky J.J., Adams M.D., Cargill M.;
    RT   ;
    RL   Submitted (16-NOV-2003) to the EMBL/GenBank/DDBJ databases.
    RL   Celera Genomics, 45 West Gude Drive, Rockville, MD 20850, USA
    XX
    CC   This sequence was made by sequencing genomic exons and ordering
    CC   them based on alignment.
    XX
    FH   Key             Location/Qualifiers
    FH
    FT   source          1..402
    FT                   /organism="Homo sapiens"
    FT                   /mol_type="genomic DNA"
    FT                   /db_xref="taxon:9606"
    FT   gene            <1..>402
    FT                   /gene="FAU"
    FT                   /locus_tag="HCM4175"
    XX
    SQ   Sequence 402 BP; 95 A; 110 C; 129 G; 68 T; 0 other;
         atgcagctct ttgtccgcgc ccaggagcta cacaccttcg aggtgaccgg ccaggaaacg        60
         gtcgcccaga tcaaggctca tgtagcctca ctggagggca ttgccccgga agatcaagtc       120
         gtgctcctgg caggcgcgcc cctggaggat gaggccactc tgggccagtg cggggtggag       180
         gccctgacta ccctggaagt agcaggccgc atgcttggag gtaaagtcca tggttccctg       240
         gcccgtgctg gaaaagtgag aggtcagact cctaaggtgg ccaaacagga gaagaagaag       300
         aagaagacag gtcgggctaa gcggcggatg cagtacaacc ggcgctttgt caacgttgtg       360
         cccacctttg gcaagaagaa gggccccaat gccaactctt aa                          402
    //

## 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.

By default diffseq writes a 'diffseq' report file.

### Output files for usage example

### File: x65923.diffseq

    ########################################
    # Program: diffseq
    # Rundate: Tue 15 Jul 2008 12:00:00
    # Commandline: diffseq
    #    [-asequence] tembl:x65923
    #    [-bsequence] tembl:ay411291
    # Report_format: diffseq
    # Report_file: x65923.diffseq
    # Additional_files: 2
    # 1: X65923.diffgff (Feature file for first sequence)
    # 2: AY411291.diffgff (Feature file for second sequence)
    ########################################

    #=======================================
    #
    # Sequence: X65923     from: 1   to: 518
    # HitCount: 1
    #
    # Compare: AY411291     from: 1   to: 402
    #
    # X65923 overlap starts at 57
    # AY411291 overlap starts at 1
    #
    #
    #=======================================

    X65923 284-284 Length: 1
    Feature: CDS 57-458 gene='fau' db_xref='GDB:135476' db_xref='GOA:P35544' db_xref='GOA:P62861' db_xref='HGNC:3597' db_xref='UniProtKB/Swiss-Prot:P35544' db_xref='UniProtKB/Swiss-Prot:P62861' protein_id='CAA46716.1'
    Feature: misc_feature 279-458 note='S30 part'
    Sequence: t
    Sequence: c
    Feature: gene 1-402 gene='FAU' locus_tag='HCM4175'
    AY411291 228-228 Length: 1

    #---------------------------------------
    #
    # Overlap_end: 458 in X65923
    # Overlap_end: 402 in AY411291
    #
    # SNP_count: 1
    # Transitions: 1
    # Transversions: 0
    #
    #---------------------------------------

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

### File: AY411291.diffgff

    ##gff-version 3
    ##sequence-region AY411291 1 402
    #!Date 2008-07-15
    #!Type DNA
    #!Source-version EMBOSS 6.0.0
    AY411291	diffseq	sequence_conflict	228	228	1.000	+	.	ID="AY411291.1";note="SNP in X65923";replace="t"

### File: X65923.diffgff

    ##gff-version 3
    ##sequence-region X65923 1 518
    #!Date 2008-07-15
    #!Type DNA
    #!Source-version EMBOSS 6.0.0
    X65923	diffseq	sequence_conflict	284	284	1.000	+	.	ID="X65923.1";note="SNP in AY411291";replace="c"

The first line is the title giving the names of the sequences used.

The next two non-blank lines state the positions in each sequence where the detected overlap between them starts.

There then follows a set of reports of the mismatches between the sequences.
Each report consists of 4 or more lines.

- The first line has the name of the first sequence followed by the start and end positions of the mismatched region in that sequence, followed by the length of the mismatched region. If the mismatched region is of zero length in this sequence, then only the position of the last matching base before the mismatch is given.
- If a feature of the first sequence overlaps with this mismatch region, then one or more lines starting with 'Feature:' comes next with the type, position and tag field of the feature.
- Next is a line starting "Sequence:" giving the sequence of the mismatch in the first sequence.

This is followed by the equivalent information for the second sequence, but in the reverse order, namely 'Sequence:' line, 'Feature:' lines and line giving the position of the mismatch in the second sequence.

At the end of the report are two non-blank lines giving the positions in each sequence where the detected overlap between them ends.

The last three lines of the report gives the counts of SNPs (defined as a change of one nucleotide to one other nucleotide, no deletions or insertions are counted, no multi-base changes are counted).

If the input sequences are nucleic acid, The counts of transitions (Pyrimide to Pyrimidine or Purine to Purine) and transversions (Pyrimidine to Purine) are also given.

It should be noted that not all features are reported.

The 'source' feature found in all EMBL/Genbank feature table entries is not reported as this covers all of the sequence and so overlaps with any difference found in that sequence and so is uninformative and irritating. It has therefore been removed from the output report.

The translation information of CDS features is often extremely long and does not add useful information to the report. It has therefore been removed from the output report.

## Data files

None

## Notes

diffseq is useful when looking for SNPs, differences between strains of an organism and anything else that requires the differences between two eseentially identical sequences to be highlighted.

Identical sequence regions are found by creating a hash table of subsequences of user-defined size (-wordsize option, which is 10 by default). Making this value larger (e.g. 20) may speed-up the program slightly, but will mean that any two differences within `wordsize` bases bases or residues of each other will be grouped as a single region of difference. This value may be made smaller to improve the resolution of nearby differences, but the program will go much slower.

The sequences can be very long; it should be possible to find differences between sequences that are Mega-bases long. If, however, you run out of memory, use a larger word size. This increases the length between mismatches that will be reported as one event. Thus a word size of 50 will report two single-base differences that are with 50 bases of each other as one mismatch.

## References

None.

## Warnings

Not all features are compared and reported. The 'source' feature found in all EMBL/Genbank feature table entries is not reported. This feature is uninformative; it covers the entire sequence and therefore would always be reported. The translation information of CDS features is often extremely long and does not add useful information to the report. It has therefore been removed from the output report.

By default diffseq finds regions of identity that are at least as long as the specified word-size. This is what's typically required when working with long overlapping nucleic acid sequences, where the non-overlapping sequence ends are less interesting. If however, you have protein sequences or short RNA sequences however, you may well be interested in differences at the very ends. The `-globaldifferences` option when set means the differences at the ends will also be reported.

## Diagnostic Error Messages

None.

## Exit status

It always exits with status 0.

## Known bugs

None.

## See also

Program name | Description
--- | ---

## Author(s)

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

## History

Written 15th Aug 2000 - Gary Williams.

18th Aug 2000 - Added writing out GFF files of the mismatched regions

## Target users

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

## Comments

None