edialign Wiki The master copies of EMBOSS documentation are available at http://emboss.open-bio.org/wiki/Appdocs on the EMBOSS Wiki. Please help by correcting and extending the Wiki pages. Function Local multiple alignment of sequences Description edialign is an EMBOSS version of the program DIALIGN2 by B. Morgenstern. It takes as input nucleic acid or protein sequences and produces as output a multiple sequence alignment. The sequences need not be similar over their complete length, since the program constructs alignments from gapfree pairs of similar segments of the sequences. Such segment pairs are referred to as "diagonals". If (possibly) coding nucleic acid sequences are to be aligned, edialign can optionally translate the compared "nucleic acid segments" to "peptide segments", or even perform comparisons at both nucleic acid and protein levels, so as to increase the sensitivity of the comparison. Algorithm For a complete explanation of the algorithm, see the references. In short : As described in our papers, the program DIALIGN constructs alignments from gapfree pairs of similar segments of the sequences. Such segment pairs are referred to as "diagonals". Every possible diagonal is given a so-called weight reflecting the degree of similarity among the two segments involved. The overall score of an alignment is then defined as the sum of weights of the diagonals it consists of and the program tries to find an alignment with maximum score -- in other words : the program tries to find a consistent collection of diagonals with maximum sum of weights. This novel scoring scheme for alignments is the basic difference between DIALIGN and other global or local alignment methods. Note that DIALIGN does not employ any kind of gap penalty. It is possible to use a threshold T for the quality of the diagonals. In this case, a diagonal is considered for alignment only if its "weight" exceeds this threshold. Regions of lower similarity are ignored. In the first version of the program (DIALIGN 1), this threshold was in many situations absolutely necessary to obtain meaningful alignments. By contrast, DIALIGN 2 should produce reasonable alignments without a threshold, i.e. with T = 0. This is the most important difference between DIALIGN 2 and the first version of the program. Nevertheless, it is still possible to use a positive threshold T to filter out regions of lower significance and to include only high scoring diagonals into the alignment. The use of overlap weights improves the sensitivity of the program if multiple sequences are aligned but it also increases the running time, especially if large numbers of sequences are aligned. By default, "overlap weights" are used if up to 35 sequences are aligned but switched off for larger data sets. If (possibly) coding nucleic acid sequences are to be aligned, DIALIGN optionally translates the compared "nucleic acid segments" to "peptide segments" according to the genetic code -- without presupposing any of the three possible reading frames, so all combinations of reading frames get checked for significant similarity. If this option is used, the similarity among segments will be assessed on the "peptide level" rather than on the "nucleic acid level". For the levels of sequence similarity, release 2.2 of DIALIGN has two additional options: * It can measure the similarity among segment pairs at both levels of similarity (nucleotide-level and peptide-level similarity). The score of a fragment is based on whatever similarity is stronger. As a result, the program can now produce mixed alignments that contain both types of fragments. Fragments with stronger similarity at the "nucleotide level" are referred to as N-fragments whereas fragments with stronger similarity a the peptide level are called P-fragments. * If the translation or mixed alignment option is used, it is possible to consider the reverse complements of segments, too. In this case, both the original segments and their reverse complements are translated and both pairs of implied "peptide segments" are compared. This option is useful if DNA sequences contain coding regions not only on the "Watson strand" but also on the "Crick strand". The score that DIALIGN assigns to a fragment is based on the probability to find a fragment of the same respective length and number of matches (or BLOSUM values, if the translation option is used) in random sequences of the same length as the input sequences. If long genomic sequences are aligned, an iterative procedure can be applied where the program first looks for fragments with strong similarity. In subsequent steps, regions between these fragments are realigned. Here, the score of a fragment is based on random occurrence in these regions between the previously aligned segment pairs. Usage Here is a sample session with edialign % edialign Local multiple alignment of sequences Input sequence set: vtest.seq Output file [vtest.edialign]: (gapped) output sequence(s) [vtest.fasta]: Go to the input files for this example Go to the output files for this example Command line arguments Local multiple alignment of sequences Version: EMBOSS:6.4.0.0 Standard (Mandatory) qualifiers: [-sequences] seqset Sequence set filename and optional format, or reference (input USA) [-outfile] outfile [*.edialign] Output file name [-outseq] seqoutall [.] (Aligned) sequence set(s) filename and optional format (output USA) Additional (Optional) qualifiers (* if not always prompted): * -nucmode menu [n] Nucleic acid sequence alignment mode (simple, translated or mixed) (Values: n (simple); nt (translation); ma (mixed alignments)) * -revcomp boolean [N] Also consider the reverse complement -overlapw selection [default (when Nseq =< 35)] By default overlap weights are used when Nseq =<35 but you can set this to 'yes' or 'no' -linkage menu [UPGMA] Clustering method to construct sequence tree (UPGMA, minimum linkage or maximum linkage) (Values: UPGMA (UPGMA); max (maximum linkage); min (minimum linkage)) -maxfragl integer [40] Maximum fragment length (Integer 0 or more) * -fragmat boolean [N] Consider only N-fragment pairs that start with two matches * -fragsim integer [4] Consider only P-fragment pairs if first amino acid or codon pair has similarity score of at least n (Integer 0 or more) -itscore boolean [N] Use iterative score -threshold float [0.0] Threshold for considering diagonal for alignment (Number 0.000 or more) Advanced (Unprompted) qualifiers: -mask boolean [N] Replace unaligned characters by stars '*' rather then putting them in lowercase -dostars boolean [N] Activate writing of stars instead of numbers -starnum integer [4] Put up to n stars '*' instead of digits 0-9 to indicate level of conservation (Integer 0 or more) Associated qualifiers: "-sequences" 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 -odirectory2 string Output directory "-outseq" associated qualifiers -osformat3 string Output seq format -osextension3 string File name extension -osname3 string Base file name -osdirectory3 string Output directory -osdbname3 string Database name to add -ossingle3 boolean Separate file for each entry -oufo3 string UFO features -offormat3 string Features format -ofname3 string Features file name -ofdirectory3 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 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 Input file format edialign reads nucleotide or protein sequences to be aligned. 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 File: vtest.seq >HTL2 LDTAPCLFSDGSPQKAAYVLWDQTILQQDITPLPSHETHSAQKGELLALICGLRAAKPWP SLNIFLDSKY >MMLV GKKLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIH CPGHQKGHSAEARGNRMADQAARKAAITETPDTSTLL >HEPB RPGLCQVFADATPTGWGLVMGHQRMRGTFSAPLPIHTAELLAACFARSRSGANIIGTDNS GRTSLYADSPSVPSHLPDRVH Output file format edialign produces two output files with a multiple sequence alignment. The first one is a file in DIALIGN format and the second one is a sequence file in any format you choose (by default fastA). 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. Capital letters denote aligned residues, i.e. residues involved in at least one of the "diagonals" in the alignment. Lower-case letters denote residues not belonging to any of these selected "diagonals". They are not considered to be aligned by DIALIGN. Thus, if a lower-case letter is standing in the same column with other letters, this is pure chance ; these residues are not considered to be homologous. Numbers below the alignment reflect the degree of local similarity among sequences. More precisely, they represent the sum of weights of fragments connecting residues at the respective position. These numbers are normalized such that regions of maximum similarity always get a score of 9 - no matter how strong this maximum simliarity is. In previous verions of the program, '*' characters were used instead of numbers ; with the -stars=n option, '*' characters can be used as previously. At the bottom of the file you can find the "guide tree" used to make the alignment, written in "nested parentheses" format. Output files for usage example File: vtest.fasta >HTL2 ldtapC-LFSDGSPQKAAYVLWDQTILQQDITPLPSHethsaqkgELLAliCglraAKPW PSLNIFLDSKY------------------------------------------------- ----------------------------------------- >MMLV gkk--------------------------------------------------------- --LNVYTDSRYafatahihgeiyrrrglltsegkeiknkdeilallkalflpkrlsiihc pghqkghsaeargnrmADQAARKAAITETPDTSTLL----- >HEPB rpgl-CqVFADATPTGWGLVMGHQRMRGTFSAPLPIHta------ELLAa-Cf---ARSR SGANIIg----------------------------------------------------- ----------------TDNSGRTSLYADSPSVPSHLpdrvh File: vtest.edialign DIALIGN 2.2.1 ************* Program code written by Burkhard Morgenstern and Said Abdeddaim e-mail contact: dialign (at) gobics (dot) de Published research assisted by DIALIGN 2 should cite: Burkhard Morgenstern (1999). DIALIGN 2: improvement of the segment-to-segment approach to multiple sequence alignment. Bioinformatics 15, 211 - 218. For more information, please visit the DIALIGN home page at http://bibiserv.techfak.uni-bielefeld.de/dialign/ ************************************************************ program call: edialign Aligned sequences: length: ================== ======= 1) HTL2 70 2) MMLV 97 3) HEPB 81 Average seq. length: 82.7 Please note that only upper-case letters are considered to be aligned. Alignment (DIALIGN format): =========================== HTL2 1 ldtapC-LFS DGSPQKAAYV LWDQTILQQD ITPLPSHeth saqkgELLAl MMLV 1 gkk------- ---------- ---------- ---------- ---------- HEPB 1 rpgl-CqVFA DATPTGWGLV MGHQRMRGTF SAPLPIHta- -----ELLAa 0000000999 9999999999 9999999999 9999999000 0000000000 HTL2 50 iCglraAKPW PSLNIFLDSK Y--------- ---------- ---------- MMLV 4 ---------- --LNVYTDSR Yafatahihg eiyrrrgllt segkeiknkd HEPB 44 -Cf---ARSR SGANIIg--- ---------- ---------- ---------- 0000000000 0077777777 7000000000 0000000000 0000000000 HTL2 71 ---------- ---------- ---------- ---------- ---------- MMLV 42 eilallkalf lpkrlsiihc pghqkghsae argnrmADQA ARKAAITETP HEPB 57 ---------- ---------- ---------- ------TDNS GRTSLYADSP 0000000000 0000000000 0000000000 0000001111 1111111111 HTL2 71 ---------- - MMLV 92 DTSTLL---- - HEPB 71 SVPSHLpdrv h 1111110000 0 Sequence tree: ============== Tree constructed using UPGMA based on DIALIGN fragment weight scores ((HTL2 :0.145587HEPB :0.145587):0.108531MMLV :0.254117); Data files The scoring schemes are hard coded in the program and cannot be changed. For proteins edialign always uses the BLOSUM62 table. Notes We strongly recommend to use the "translation" option if nucleic acid sequences are expected to contain protein coding regions, as it will significantly increase the sensitivity of the alignment procedure in such cases. If you want to compare long genomic sequences it is recommended to speed up the algorithm by: * setting "Nucleic acid sequence alignment mode" to "mixed alignment" (-nucmode=ma) * setting "Maximum fragment length" to 30 (-lmax=30) * setting "Consider only N-fragment pairs that start with two matches" to yes (-fragmat) and setting the similarity score threshold for considering P-fragment pairs to 8 (-fragsim=8) (which actually implies that you consider only fragments that start with a match). * setting the "Threshold" T to 2.0 (-threshold=2.0) It is also recommended to increase the chance of finding coding exons by setting "Nucleic acid sequence alignment mode" to "mixed alignment" (-nucmode=ma) and setting "Also consider the reverse complement" (-revcomp). References 1. B. Morgenstern, A. Dress, T. Werner. Multiple DNA and protein sequence alignment based on segment-to-segment comparison. Proc. Natl. Acad. Sci. USA 93, 12098 - 12103 (1996) 2. B. Morgenstern. DIALIGN 2: improvement of the segment-to-segment approach to multiple sequence alignment. Bioinformatics 15, 211 - 218 (1999). 3. B. Morgenstern, O. Rinner, S. Abdeddaim, D. Haase, K. F. X. Mayer, A. W. M. Dress H.-W. Mewes. Exon discovery by genomic sequence alignment. Bioinformatics 18, 777 - 787 (2002) Warnings Remember that lowercase characters represent parts of the sequence that are not aligned. You should not use the dialign output as such for sequence family or phylogeny studies, but take only part of the alignment and/or remove the lowercase characters using a multiple sequence editor. The current version of the program has no provision for doing this automatically. Diagnostic Error Messages None. Exit status It always exits with status 0. Known bugs None. See also Program name Description emma Multiple sequence alignment (ClustalW wrapper) infoalign Display basic information about a multiple sequence alignment plotcon Plot conservation of a sequence alignment prettyplot Draw a sequence alignment with pretty formatting showalign Display a multiple sequence alignment in pretty format tranalign Generate an alignment of nucleic coding regions from aligned proteins Author(s) The EMBOSS direct port was done 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 (c) emboss.open-bio.org) not to the original author. based on ACD written by Guy Bottu (gbottu@ben.vub.ac.be) for a wrapper written at BEN, ULB, Brussels, Belgium The program DIALIGN itself was written by Burkhard Morgenstern, Said Abdeddaim, Klaus Hahn, Thomas Werner, Kornelie Frech and Andreas Dress. Universitaet Bielefeld (FSPM and International Graduate School in Bioinformatics and Genome Research) - GSF Research Center (ISG, IBB, MIPS/IBI) - North Carolina State University - Universite de Rouen - MPI fuer Biochemie (Martinsried) - University of Goettingen, Institute of Microbiology and Genetics - Rhone-Poulenc Rorer For help on the original DIALIGN2, contact: dialign@gobics.de History First committed on 5th December 2006. Target users This program is intended to be used by everyone and everything, from naive users to embedded scripts. Comments None