BACKGROUND:



The models developed to characterize the evolution of multigene families (such as the birth-and-death and the concerted models) have also been applied on the level of sequence repeats inside a gene/protein. Phylogenetic reconstruction is the method of choice to study the evolution of gene families and also sequence repeats in the light of these models. The characterization of the gene family evolution in view of the evolutionary models is done by the evaluation of the clustering of the sequences with the originating loci in mind. As the locus represents positional information, it is straightforward that in the case of the repeats the exact position in the sequence should be used, as the simple numbering according to repeat order can be misleading.

RESULTS:



We have developed a novel rapid visual approach to study repeat evolution, that takes into account the exact repeat position in a sequence. The "pairwise repeat homology diagram" visualizes sequence repeats detected by a profile HMM in a pair of sequences and highlights their homology relations inferred by a phylogenetic tree. The method is implemented in a Perl script (t2prhd) available for downloading at http://t2prhd.sourceforge.net and is also accessible as an online tool at http://t2prhd.brc.hu. The power of the method is demonstrated on the EGF-like and fibronectin-III-like (Fn-III) domain repeats of three selected mammalian Tenascin sequences.

CONCLUSION:



Although pairwise repeat homology diagrams do not carry all the information provided by the phylogenetic tree, they allow a rapid and intuitive assessment of repeat evolution. We believe, that t2prhd is a helpful tool with which to study the pattern of repeat evolution. This method can be particularly useful in cases of large datasets (such as large gene families), as the command line interface makes it possible to automate the generation of pairwise repeat homology diagrams with the aid of script

A Carmon

AL Hughes

BE Galindo

Botond Sipos

C Cvitanich

C Letondal

D Liao

H Johannesson

H Quesada

István Andó

JD Thompson

JE Stajich

Kálmán Somogyi

M Nei

M Paoletti

MJ Kwakkenbosi

N Frankel

S Guindon

T Meeds

WJ Swanson

Zsolt Pénzes

English

PubMed

Sipos, Botond

Somogyi, Kálmán

Andó, István

Pénzes, Zsolt

Repository of the Academy's Library

BioMed CentralBMC BioinformaticsssOpen AcceMethodology articlet2prhd: a tool to study the patterns of repeat evolutionBotond Sipos*1, Kálmán Somogyi1, István Andó1 and Zsolt Pénzes1,2Address: 1Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary and 2Department of Ecology, University of Szeged, Egyetem street 2, H-6721, Szeged, HungaryEmail: Botond Sipos* - sbotond@gmail.com; Kálmán Somogyi - somogyi@brc.hu; István Andó - ando@brc.hu; Zsolt Pénzes - penzes@bio.u-szeged.hu* Corresponding author    AbstractBackground: The models developed to characterize the evolution of multigene families (such asthe birth-and-death and the concerted models) have also been applied on the level of sequencerepeats inside a gene/protein. Phylogenetic reconstruction is the method of choice to study theevolution of gene families and also sequence repeats in the light of these models. Thecharacterization of the gene family evolution in view of the evolutionary models is done by theevaluation of the clustering of the sequences with the originating loci in mind. As the locusrepresents positional information, it is straightforward that in the case of the repeats the exactposition in the sequence should be used, as the simple numbering according to repeat order canbe misleading.Results: We have developed a novel rapid visual approach to study repeat evolution, that takesinto account the exact repeat position in a sequence. The "pairwise repeat homology diagram"visualizes sequence repeats detected by a profile HMM in a pair of sequences and highlights theirhomology relations inferred by a phylogenetic tree. The method is implemented in a Perl script(t2prhd) available for downloading at http://t2prhd.sourceforge.net and is also accessible as anonline tool at http://t2prhd.brc.hu. The power of the method is demonstrated on the EGF-like andfibronectin-III-like (Fn-III) domain repeats of three selected mammalian Tenascin sequences.Conclusion: Although pairwise repeat homology diagrams do not carry all the informationprovided by the phylogenetic tree, they allow a rapid and intuitive assessment of repeat evolution.We believe, that t2prhd is a helpful tool with which to study the pattern of repeat evolution. Thismethod can be particularly useful in cases of large datasets (such as large gene families), as thecommand line interface makes it possible to automate the generation of pairwise repeat homologydiagrams with the aid of scripts.BackgroundThe conceptual models developed to explain the evolu-tion of multigene families [1,2] have also been applied tosequence repeats inside a gene/protein. Studies of repeatevolution [3-9] have often revealed complex patterns:some repeats may evolve in concert, subject to homogeni-zation, while other repeats may maintain their sequenceidentity, which is more consistent with birth-and-deathand/or divergent evolution. Phylogenetic reconstructionis an established approach with which to study the modePublished: 18 January 2008BMC Bioinformatics 2008, 9:27 doi:10.1186/1471-2105-9-27Received: 22 August 2007Accepted: 18 January 2008This article is available from: http://www.biomedcentral.com/1471-2105/9/27© 2008 Sipos et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 5(page number not for citation purposes)BMC Bioinformatics 2008, 9:27 http://www.biomedcentral.com/1471-2105/9/27of evolution of multigene families [10-14] and repeats [3-9]. In the analysis of closely related species, the associa-tion of genes on the same locus (orthologues) on phylo-genetic trees suggests independent evolution of therespective members, as expected for the birth-and-deathmodel. On the contrary, the association of paraloguesfrom the same species suggests homogenization, which isconsistent with the concerted model. The application ofthe same logic to repeats poses a number of problems.Studies on the evolution of multigene families by phylo-genetic methods make use of a prior concept of homologydefined by the locus. As the term "locus" denotes genomicposition, it follows that the exact repeat position in thefull sequence (starting and ending positions) should beused to identify them. This approach has seldom beenused in the previous studies, probably because it compli-cates the interpretation of the phylogenetic trees. Instead,a simple numbering according to repeat order is oftenapplied, although this can be misleading: if a repeat is notdetected by the selected method (for example the profileHidden Markov Model (HMM) method) in one of thecompared sequences, the ordinal numbers of the follow-ing repeats will be shifted by one and hence the analysiswill become laborious.Results and DiscussionTo simplify the analyses of repeat evolution, a novel rapidvisual method has been developed to highlight the rela-tionships of repeats detected in a pair of sequences by theprofile HMM method. The primary aim of the applicationis the analysis of tandem amino acid sequence repeats butDNA sequences can also be used with a profile HMMtrained with DNA sequences. When DNA sequences areused, the script does not consider the reverse complementof the sequences, so the usefulness of this application in agenomic context (for example in the case of transportableelements) might be limited, though the implementationcan be easily extended to handle this kind of analysis.The "pairwise repeat homology diagram" visualizesrepeats in a simple scheme, together with their homologyrelations (orthology and paralogy) inferred by a phyloge-netic tree, providing an intuitive way to analyse the pat-terns of repeat evolution. This visualization also facilitatesa survey of the sequence structure (size, linker sequencesand non-repetitive regions). Regions in which consecutiverepeats are connected with those in the other sequenceforming a ladder-like pattern are most likely to haveevolved independently following the birth-and-deathand/or divergent process. On the contrary, in regionswhere repeats have only internal or no identified connec-tions, repeats possibly evolve in concert. The patternsdemonstrated by internal connections can reveal units ofconcerted evolution or recent internal duplications. Itshould be taken into account, however, that if there areclades formed by more than two identical sequences, thebranching pattern of such clades, and hence the identifiedrelation, is arbitrary and uninformative in this kind ofanalysis.ImplementationThe method is implemented in a Perl script with com-mand line interface (t2prhd, standing for "tree to pairwiserepeat homology diagram"). The repeats are first identi-fied with hmmsearch from the HMMER package [15] and aprofile HMM specified by the user. Raw search results areparsed by using BioPerl [16] modules and the extractedrepeats are aligned with hmmalign. The resulting align-ment is converted into Fasta and sequencial PHYLIP for-mats and a phylogenetic tree is built by using CLUSTAL W[17] (with default parameters) or by using PhyML [18].The script reads in the resulting tree as a Bio::Tree::TreeIobject. After getting the list of all leaf nodes it finds the"sister leaf nodes" (leaf node pairs having the same ances-tor) by an algorithm that for n leaf nodes (repeats) needsin the best case  and in the worst case n iterations. So,the asymptotic upper bound for the time complexity ofthis algorithm is O(n) and the asymptotic lower bound isΩ( ). These sister leaf nodes have a most recent commonancestor as indicated by the tree and accordingly weregard them as unambiguously identified homologues.The script creates an SVG (Scalable Vector Graphics) filecontaining the diagram by using the XML::Writer moduleand saves the outputs and logs produced by the externalapplications. Optionally, it can also generate a LaTeX out-put. The identified homology relations are represented bylines or arcs connecting the respective repeats: blue linesare drawn between repeats from different sequences(orthology), and brown arcs in cases of internal relations(paralogy). The colour intensity of the connecting lines isa function of the patristic distance between the respectiveleaf nodes (d) divided by the total tree length (T):. The default value of the "colour gradientparameter" w is 1 (linear colour scale), but by setting thisparameter the colour scale can be tuned so that one candiscriminate between close distance values. To make theinterpretation of the colour scale easier a legend is drawn.The generated SVG file can be viewed by Firefox 1.5 [19] orhigher as an example and can be rasterized (and alsoviewed) using the Batik toolkit [20] or any other imageeditor capable to handle the SVG format. The generatedn2n21 −( )dT wPage 2 of 5(page number not for citation purposes)BMC Bioinformatics 2008, 9:27 http://www.biomedcentral.com/1471-2105/9/27LaTeX file should be processed by pdflatex, the pgf/TikZ,fancyhdr, xcolor and fullpage packages are required. Thescript has a manual page embedded in POD format.The script is also accessible as an online tool [21] (withCLUSTAL W back-end only) through a web interface cre-ated by using the Pise form generator [22]. The real powerof this visual method is manifested in studies with largedata sets, where the analysis of numerous or large treeswould be highly laborious. In these cases, it is very advan-tageous that the command line interface enables the useof the scripts to automate the diagram generation.ExampleTenascins are extracellular matrix glycoproteins contain-ing regions of repeated EGF-like and fibronectin-III-like(Fn-III) domains. The evolution of these repeateddomains in mammalian Tenascins has been studied indetail by means of phylogenetic and other methods [8].To illustrate the power of the method, we generated pair-wise repeat homology diagrams with three selected pro-tein sequences. The protein sequences corresponding tothe DNA sequences studied by Hughes [8] were used(abbreviations and GenBank accession numbers in paren-theses): human Tenascin X (TXH, [GenBank:AAB47488.1]), human Tenascin C (TCH, [GenBank:CAA39628.1]) and murine Tenascin X (TXM, [GenBank:AAB82015.1]). The profile HMM files were downloadedfrom Pfam (EGF-like: PF00008.17, Fn-III: PF00041.11).Hughes [8] concluded that EGF domain repeats under-went homogenization within each Tenascin gene afterduplication, but remained conserved after the divergenceof rodents and primates. The same conclusions can bedrawn after the evaluation of the diagrams generated withparalogous (Figure 1A and 1B) and orthologous (Figure1C) sequence pairs. Our diagrams are also in accordancewith the conclusions of Hughes regarding the evolution ofPairwise repeat homology diagrams of selected Tenascin proteins (SVG outputs)Figure 1Pairwise repeat homology diagrams of selected Tenascin proteins (SVG outputs). The diagrams were produced with the default PhyML parameters of the script (WAG+Γ+I, 4 gamma categories, gamma parameter and proportion of invari-able sites estimated by ML, BIONJ starting tree) and with colour gradient paramater w set to 2. The repeats (EGF on A, B and C; Fn-III on A', B' and C') are indicated by red rectangles in the protein sequence schemes (N-termini on top). The identified homology relations are represented by lines or arcs connecting the respective repeats (blue lines between repeats of different protein sequences (orthology), and brown arcs in case of internal relations (paralogy)). The colour scale bar demonstrates the colour intensities as a function of the patristic distance between the respective clades divided by the total tree length. The lines represent the values between 0 and 1 by units of 0.1. The sequence positions are shown on the right of each scheme. Abbrevi-ations: TCH: Homo sapiens Tenascin C; TXH: Homo sapiens Tenascin X and TXM: Mus musculus Tenascin X.Page 3 of 5(page number not for citation purposes)BMC Bioinformatics 2008, 9:27 http://www.biomedcentral.com/1471-2105/9/27Fn-III type domain repeats. These repeats can be dividedinto three categories. The last three C-terminal repeatsdemonstrate conservation since the duplication of theTenascin X and Tenascin C genes (Figure 1A', B' and 1C').Other repeats became homogenized within each genesubsequent to gene duplication, but have remained con-served following the divergence of primates and rodents(Figure 1C'). The repeats of the third category haveevolved in a concerted fashion in rodent and primate lin-eages since their divergence (Figure 1C').ConclusionAlthough pairwise repeat homology diagrams do notcarry all the information about the phylogenetic tree onwhich they are based, by visualizing the exact positions ofthe repeats and the homology relations, they permit arapid and intuitive assessment of the patterns of repeatevolution (compare Figure 1C' with Figure 2). These fea-tures make t2prhd a powerful tool, especially in cases ofmassive datasets, as in studies of repeat evolution in largegene families.Availability and requirements• Project name: t2prhd• Project home page: http://t2prhd.sourceforge.net• Online access: http://t2prhd.brc.hu• Operating system(s): OS Independent (Written in aninterpreted language)• Programming language: Perl• Other requirements: Perl version 5.8.8 or higher withthe standard modules, BioPerl modules (version 1.4.0 orhigher), XML::Writer module, HMMER package 2.3.2 orhigher, CLUSTAL W version 1.83 and/or PhyML version2.4.4 or higher• License: GNU General Public License• Any restrictions to use by non-academics: noneAuthors' contributionsBS, KS and ZP developed the approach. BS wrote the pro-gram and the manual and created the website. ZPreviewed the code and the manual and tested the programfunctionality. BS and KS wrote the manuscript. IA and ZPconceived and coordinated the project and refined themanuscript.Phylogenetic tree of Fn-III repeats of Tenascin X proteins of Homo sapiens and Mus musculusFigure 2Phylogenetic tree of Fn-III repeats of Tenascin X pro-teins of Homo sapiens and Mus musculus. Maximum Likelihood tree produced during the analysis of human versus murine Tenascin X sequences with Fn-III profile HMM (dia-gram on Figure 1C'). Repeats are identified by starting and ending positions. Abbreviations: see Figure 1.Page 4 of 5(page number not for citation purposes)BMC Bioinformatics 2008, 9:27 http://www.biomedcentral.com/1471-2105/9/27Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralAcknowledgementsJoint doctoral fellowship of The National Centre for Scholarships Abroad of the Romanian Ministry of Education and Research and the Hungarian Scholarship Board to BS; FEBS Long-term Fellowship to KS; Bolyai Research Scholarship of the Hungarian Academy of Sciences to ZP. This work was supported by the Hungarian Scientific Research Fund grant (NI60442). Fur-ther support for the IT infrastructure was provided by the OMAI DT-D/4/005/2006 and BIO-00126 funds of the Hungarian Ministry of Education. We would like to thank for the comments and suggestions to the anonymous reviewers, for useful discussion on the source code to Szilárd Páll and for the assistance in online hosting to Ernő Homolya.References1. Nei M, Rooneyi AP: Concerted and birth-and-death evolutionof multigene families.  Annu Rev Genet 2005, 39:121-52.2. Liao D: Concerted evolution: molecular mechanism and bio-logical implications.  Am J Hum Genet 1999, 64:24-30.3. Paoletti M, Saupe SJ, Clavé C: Genesis of a fungal non-self recog-nition repertoire.  PLoS ONE 2007, 2:e283.4. Carmon A, M W, Hassan J, Baron M, Macintyre R: Concerted evo-lution within the Drosophila dumpy gene.  Genetics 2007,176:309-25.5. Johannesson H, Townsend JP, Hung CY, Cole GT, Taylor JW: Con-certed evolution in the repeats of an immunomodulating cellsurface protein, SOWgp, of the human pathogenic fungiCoccidioides immitis and C. posadasii.  Genetics 2005, 171:109-17.6. Galindo BE, Vacquier VD, Swanson WJ: Positive selection in theegg receptor for abalone sperm lysin.  Proc Natl Acad Sci USA2003, 100:4639-43.7. Meeds T, Lockard E, Livingston BT: Special evolutionary proper-ties of genes encoding a protein with a simple amino acidrepeat.  J Mol Evol 2001, 53:180-90.8. Hughes AL: Concerted evolution of exons and introns in theMHC-linked tenascin-X gene of mammals.  Mol Biol Evol 1999,16:1558-67.9. Swanson WJ, Vacquier VD: Concerted evolution in an eggreceptor for a rapidly evolving abalone sperm protein.  Sci-ence 1998, 281:710-2.10. Cvitanich C, Salcido M, Judelson HS: Concerted evolution of atandemly arrayed family of mating-specific genes in Phy-tophthora analyzed through inter- and intraspecific compar-isons.  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Concerted evolution within the Drosophila dumpy gene. Genetics

Concerted evolution: molecular mechanism and biological implications.

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HS: Concerted evolution of a tandemly arrayed family of mating-specific genes in Phytophthora analyzed through inter- and intraspecific comparisons. Mol Genet Genomics

Iusem ND: Evolutionary history of the Asr gene family. Gene

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Rooneyi AP: Concerted and birth-and-death evolution of multigene families. Annu Rev Genet

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T2prhd: a tool to study the patterns of repeat evolution

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t2prhd: a tool to study the patterns of repeat evolution

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BioMed CentralBMC Bioinformatics
ssOpen AcceMethodology article
t2prhd: a tool to study the patterns of repeat evolution
Botond Sipos*1, Kálmán Somogyi1, István Andó1 and Zsolt Pénzes1,2
Address: 1Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, P.O. Box 521, H-6701 Szeged, Hungary and 
2Department of Ecology, University of Szeged, Egyetem street 2, H-6721, Szeged, Hungary
Email: Botond Sipos* - sbotond@gmail.com; Kálmán Somogyi - somogyi@brc.hu; István Andó - ando@brc.hu; Zsolt Pénzes - penzes@bio.u-
szeged.hu
* Corresponding author    
Abstract
Background: The models developed to characterize the evolution of multigene families (such as
the birth-and-death and the concerted models) have also been applied on the level of sequence
repeats inside a gene/protein. Phylogenetic reconstruction is the method of choice to study the
evolution of gene families and also sequence repeats in the light of these models. The
characterization of the gene family evolution in view of the evolutionary models is done by the
evaluation of the clustering of the sequences with the originating loci in mind. As the locus
represents positional information, it is straightforward that in the case of the repeats the exact
position in the sequence should be used, as the simple numbering according to repeat order can
be misleading.
Results: We have developed a novel rapid visual approach to study repeat evolution, that takes
into account the exact repeat position in a sequence. The "pairwise repeat homology diagram"
visualizes sequence repeats detected by a profile HMM in a pair of sequences and highlights their
homology relations inferred by a phylogenetic tree. The method is implemented in a Perl script
(t2prhd) available for downloading at http://t2prhd.sourceforge.net and is also accessible as an
online tool at http://t2prhd.brc.hu. The power of the method is demonstrated on the EGF-like and
fibronectin-III-like (Fn-III) domain repeats of three selected mammalian Tenascin sequences.
Conclusion: Although pairwise repeat homology diagrams do not carry all the information
provided by the phylogenetic tree, they allow a rapid and intuitive assessment of repeat evolution.
We believe, that t2prhd is a helpful tool with which to study the pattern of repeat evolution. This
method can be particularly useful in cases of large datasets (such as large gene families), as the
command line interface makes it possible to automate the generation of pairwise repeat homology
diagrams with the aid of scripts.
Background
The conceptual models developed to explain the evolu-
tion of multigene families [1,2] have also been applied to
sequence repeats inside a gene/protein. Studies of repeat
evolution [3-9] have often revealed complex patterns:
some repeats may evolve in concert, subject to homogeni-
zation, while other repeats may maintain their sequence
identity, which is more consistent with birth-and-death
and/or divergent evolution. Phylogenetic reconstruction
is an established approach with which to study the mode
Published: 18 January 2008
BMC Bioinformatics 2008, 9:27 doi:10.1186/1471-2105-9-27
Received: 22 August 2007
Accepted: 18 January 2008
This article is available from: http://www.biomedcentral.com/1471-2105/9/27
© 2008 Sipos et al; licensee BioMed Central Ltd. 
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), 
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 5
(page number not for citation purposes)
BMC Bioinformatics 2008, 9:27 http://www.biomedcentral.com/1471-2105/9/27of evolution of multigene families [10-14] and repeats [3-
9]. In the analysis of closely related species, the associa-
tion of genes on the same locus (orthologues) on phylo-
genetic trees suggests independent evolution of the
respective members, as expected for the birth-and-death
model. On the contrary, the association of paralogues
from the same species suggests homogenization, which is
consistent with the concerted model. The application of
the same logic to repeats poses a number of problems.
Studies on the evolution of multigene families by phylo-
genetic methods make use of a prior concept of homology
defined by the locus. As the term "locus" denotes genomic
position, it follows that the exact repeat position in the
full sequence (starting and ending positions) should be
used to identify them. This approach has seldom been
used in the previous studies, probably because it compli-
cates the interpretation of the phylogenetic trees. Instead,
a simple numbering according to repeat order is often
applied, although this can be misleading: if a repeat is not
detected by the selected method (for example the profile
Hidden Markov Model (HMM) method) in one of the
compared sequences, the ordinal numbers of the follow-
ing repeats will be shifted by one and hence the analysis
will become laborious.
Results and Discussion
To simplify the analyses of repeat evolution, a novel rapid
visual method has been developed to highlight the rela-
tionships of repeats detected in a pair of sequences by the
profile HMM method. The primary aim of the application
is the analysis of tandem amino acid sequence repeats but
DNA sequences can also be used with a profile HMM
trained with DNA sequences. When DNA sequences are
used, the script does not consider the reverse complement
of the sequences, so the usefulness of this application in a
genomic context (for example in the case of transportable
elements) might be limited, though the implementation
can be easily extended to handle this kind of analysis.
The "pairwise repeat homology diagram" visualizes
repeats in a simple scheme, together with their homology
relations (orthology and paralogy) inferred by a phyloge-
netic tree, providing an intuitive way to analyse the pat-
terns of repeat evolution. This visualization also facilitates
a survey of the sequence structure (size, linker sequences
and non-repetitive regions). Regions in which consecutive
repeats are connected with those in the other sequence
forming a ladder-like pattern are most likely to have
evolved independently following the birth-and-death
and/or divergent process. On the contrary, in regions
where repeats have only internal or no identified connec-
tions, repeats possibly evolve in concert. The patterns
demonstrated by internal connections can reveal units of
concerted evolution or recent internal duplications. It
should be taken into account, however, that if there are
clades formed by more than two identical sequences, the
branching pattern of such clades, and hence the identified
relation, is arbitrary and uninformative in this kind of
analysis.
Implementation
The method is implemented in a Perl script with com-
mand line interface (t2prhd, standing for "tree to pairwise
repeat homology diagram"). The repeats are first identi-
fied with hmmsearch from the HMMER package [15] and a
profile HMM specified by the user. Raw search results are
parsed by using BioPerl [16] modules and the extracted
repeats are aligned with hmmalign. The resulting align-
ment is converted into Fasta and sequencial PHYLIP for-
mats and a phylogenetic tree is built by using CLUSTAL W
[17] (with default parameters) or by using PhyML [18].
The script reads in the resulting tree as a Bio::Tree::TreeI
object. After getting the list of all leaf nodes it finds the
"sister leaf nodes" (leaf node pairs having the same ances-
tor) by an algorithm that for n leaf nodes (repeats) needs
in the best case  and in the worst case n iterations. So,
the asymptotic upper bound for the time complexity of
this algorithm is O(n) and the asymptotic lower bound is
Ω( ). These sister leaf nodes have a most recent common
ancestor as indicated by the tree and accordingly we
regard them as unambiguously identified homologues.
The script creates an SVG (Scalable Vector Graphics) file
containing the diagram by using the XML::Writer module
and saves the outputs and logs produced by the external
applications. Optionally, it can also generate a LaTeX out-
put. The identified homology relations are represented by
lines or arcs connecting the respective repeats: blue lines
are drawn between repeats from different sequences
(orthology), and brown arcs in cases of internal relations
(paralogy). The colour intensity of the connecting lines is
a function of the patristic distance between the respective
leaf nodes (d) divided by the total tree length (T):
. The default value of the "colour gradient
parameter" w is 1 (linear colour scale), but by setting this
parameter the colour scale can be tuned so that one can
discriminate between close distance values. To make the
interpretation of the colour scale easier a legend is drawn.
The generated SVG file can be viewed by Firefox 1.5 [19] or
higher as an example and can be rasterized (and also
viewed) using the Batik toolkit [20] or any other image
editor capable to handle the SVG format. The generated
n
2
n
2
1 −( )dT
wPage 2 of 5
(page number not for citation purposes)
BMC Bioinformatics 2008, 9:27 http://www.biomedcentral.com/1471-2105/9/27LaTeX file should be processed by pdflatex, the pgf/TikZ,
fancyhdr, xcolor and fullpage packages are required. The
script has a manual page embedded in POD format.
The script is also accessible as an online tool [21] (with
CLUSTAL W back-end only) through a web interface cre-
ated by using the Pise form generator [22]. The real power
of this visual method is manifested in studies with large
data sets, where the analysis of numerous or large trees
would be highly laborious. In these cases, it is very advan-
tageous that the command line interface enables the use
of the scripts to automate the diagram generation.
Example
Tenascins are extracellular matrix glycoproteins contain-
ing regions of repeated EGF-like and fibronectin-III-like
(Fn-III) domains. The evolution of these repeated
domains in mammalian Tenascins has been studied in
detail by means of phylogenetic and other methods [8].
To illustrate the power of the method, we generated pair-
wise repeat homology diagrams with three selected pro-
tein sequences. The protein sequences corresponding to
the DNA sequences studied by Hughes [8] were used
(abbreviations and GenBank accession numbers in paren-
theses): human Tenascin X (TXH, [GenBank:
AAB47488.1]), human Tenascin C (TCH, [GenBank:
CAA39628.1]) and murine Tenascin X (TXM, [GenBank:
AAB82015.1]). The profile HMM files were downloaded
from Pfam (EGF-like: PF00008.17, Fn-III: PF00041.11).
Hughes [8] concluded that EGF domain repeats under-
went homogenization within each Tenascin gene after
duplication, but remained conserved after the divergence
of rodents and primates. The same conclusions can be
drawn after the evaluation of the diagrams generated with
paralogous (Figure 1A and 1B) and orthologous (Figure
1C) sequence pairs. Our diagrams are also in accordance
with the conclusions of Hughes regarding the evolution of
Pairwise repeat homology diagrams of selected Tenascin proteins (SVG outputs)Figure 1
Pairwise repeat homology diagrams of selected Tenascin proteins (SVG outputs). The diagrams were produced 
with the default PhyML parameters of the script (WAG+Γ+I, 4 gamma categories, gamma parameter and proportion of invari-
able sites estimated by ML, BIONJ starting tree) and with colour gradient paramater w set to 2. The repeats (EGF on A, B and 
C; Fn-III on A', B' and C') are indicated by red rectangles in the protein sequence schemes (N-termini on top). The identified 
homology relations are represented by lines or arcs connecting the respective repeats (blue lines between repeats of different 
protein sequences (orthology), and brown arcs in case of internal relations (paralogy)). The colour scale bar demonstrates the 
colour intensities as a function of the patristic distance between the respective clades divided by the total tree length. The lines 
represent the values between 0 and 1 by units of 0.1. The sequence positions are shown on the right of each scheme. Abbrevi-
ations: TCH: Homo sapiens Tenascin C; TXH: Homo sapiens Tenascin X and TXM: Mus musculus Tenascin X.Page 3 of 5
(page number not for citation purposes)
BMC Bioinformatics 2008, 9:27 http://www.biomedcentral.com/1471-2105/9/27Fn-III type domain repeats. These repeats can be divided
into three categories. The last three C-terminal repeats
demonstrate conservation since the duplication of the
Tenascin X and Tenascin C genes (Figure 1A', B' and 1C').
Other repeats became homogenized within each gene
subsequent to gene duplication, but have remained con-
served following the divergence of primates and rodents
(Figure 1C'). The repeats of the third category have
evolved in a concerted fashion in rodent and primate lin-
eages since their divergence (Figure 1C').
Conclusion
Although pairwise repeat homology diagrams do not
carry all the information about the phylogenetic tree on
which they are based, by visualizing the exact positions of
the repeats and the homology relations, they permit a
rapid and intuitive assessment of the patterns of repeat
evolution (compare Figure 1C' with Figure 2). These fea-
tures make t2prhd a powerful tool, especially in cases of
massive datasets, as in studies of repeat evolution in large
gene families.
Availability and requirements
• Project name: t2prhd
• Project home page: http://t2prhd.sourceforge.net
• Online access: http://t2prhd.brc.hu
• Operating system(s): OS Independent (Written in an
interpreted language)
• Programming language: Perl
• Other requirements: Perl version 5.8.8 or higher with
the standard modules, BioPerl modules (version 1.4.0 or
higher), XML::Writer module, HMMER package 2.3.2 or
higher, CLUSTAL W version 1.83 and/or PhyML version
2.4.4 or higher
• License: GNU General Public License
• Any restrictions to use by non-academics: none
Authors' contributions
BS, KS and ZP developed the approach. BS wrote the pro-
gram and the manual and created the website. ZP
reviewed the code and the manual and tested the program
functionality. BS and KS wrote the manuscript. IA and ZP
conceived and coordinated the project and refined the
manuscript.
Phylogenetic tree of Fn-III repeats of Tenascin X proteins of Homo sapiens and Mus musculusFigure 2
Phylogenetic tree of Fn-III repeats of Tenascin X pro-
teins of Homo sapiens and Mus musculus. Maximum 
Likelihood tree produced during the analysis of human versus 
murine Tenascin X sequences with Fn-III profile HMM (dia-
gram on Figure 1C'). Repeats are identified by starting and 
ending positions. Abbreviations: see Figure 1.Page 4 of 5
(page number not for citation purposes)
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Acknowledgements
Joint doctoral fellowship of The National Centre for Scholarships Abroad 
of the Romanian Ministry of Education and Research and the Hungarian 
Scholarship Board to BS; FEBS Long-term Fellowship to KS; Bolyai Research 
Scholarship of the Hungarian Academy of Sciences to ZP. This work was 
supported by the Hungarian Scientific Research Fund grant (NI60442). Fur-
ther support for the IT infrastructure was provided by the OMAI DT-D/4/
005/2006 and BIO-00126 funds of the Hungarian Ministry of Education. We 
would like to thank for the comments and suggestions to the anonymous 
reviewers, for useful discussion on the source code to Szilárd Páll and for 
the assistance in online hosting to Ernő Homolya.
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Abstract Background The models developed to characterize the evolution of multigene families (such as the birth-and-death and the concerted models) have also been applied on the level of sequence repeats inside a gene/protein. Phylogenetic reconstruction is the method of choice to study the evolution of gene families and also sequence repeats in the light of these models. The characterization of the gene family evolution in view of the evolutionary models is done by the evaluation of the clustering of the sequences with the originating loci in mind. As the locus represents positional information, it is straightforward that in the case of the repeats the exact position in the sequence should be used, as the simple numbering according to repeat order can be misleading. Results We have developed a novel rapid visual approach to study repeat evolution, that takes into account the exact repeat position in a sequence. The "pairwise repeat homology diagram" visualizes sequence repeats detected by a profile HMM in a pair of sequences and highlights their homology relations inferred by a phylogenetic tree. The method is implemented in a Perl script (t2prhd) available for downloading at http://t2prhd.sourceforge.net and is also accessible as an online tool at http://t2prhd.brc.hu. The power of the method is demonstrated on the EGF-like and fibronectin-III-like (Fn-III) domain repeats of three selected mammalian Tenascin sequences. Conclusion Although pairwise repeat homology diagrams do not carry all the information provided by the phylogenetic tree, they allow a rapid and intuitive assessment of repeat evolution. We believe, that t2prhd is a helpful tool with which to study the pattern of repeat evolution. This method can be particularly useful in cases of large datasets (such as large gene families), as the command line interface makes it possible to automate the generation of pairwise repeat homology diagrams with the aid of scripts.</p

Pénzes Zsolt

Andó István

Somogyi Kálmán

Sipos Botond

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t2prhd: a tool to study the patterns of repeat evolution

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