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1. describes information to be delivered upon click Gene Information window Toggling checkboxes in the Genome Display panel lower center modifies the display of all rendered genomic segments The range of the displayed region may be easily changed in the Range Around Context Segment panel lower right hand corner Seitzer et al BMC Bioinformatics 2013 14 18 http www biomedcentral com 1471 2105 14 18 Page 4 of 8 Gena Grouping grouped together Term Definition Example Il signifi nomic elemen nomicall Genomic Context All sign cant genomic elements genomically close m am to a gene of interest aaa A set of genes in a single organism that are group 1 group 3 Genomic Working The set of genomes or genome fragments working set Set under investigation So0 gt genomic working set The combination of many non overlapping a yee A y Context Set gene groupings in all genomes in the genomic ap A Gene Cluster Context Tree All genes across all species in the genomic working yx i set that are highly homologous to one another en Multidendrogram computed from a set of gene groupings Figure 2 JContextExplorer technical terminology Technical terms relevant to the JContextExplorer program column 1 are defined column 2 and demonstrated via graphical representation column 3 gene grouping 1 gene grouping 2 gene grouping 3 gene grouping 4 file list2. Ivanova N Hooper SD Markowitz VM Kyrpides NC Gene context analysis in the Integrated Microbial Genomes IMG data management system PLoS One 2009 4 e7979 Proost S Van Bel M Sterck L Billiau K Van Parys T Van de Peer Y Vandepoele K PLAZA a comparative genomics resource to study gene and genome evolution in plants Plant Cell 2009 21 3718 3731 Muffato M Louis A Poisnel C E Roest Crollius H Genomicus a database and a browser to study gene synteny in modern and ancestral genomes Bioinformatics Oxford England 2010 26 1119 1121 Karolchik D Baertsch R Diekhans M Furey TS Hinrichs A Lu YT Roskin KM et al The UCSC genome browser database Nucleic Acids Res 2003 31 51 54 Bare JC Koide T Reiss DJ Tenenbaum D Baliga NS Integration and visualization of systems biology data in context of the genome BMC Bioinforma 2010 11 382 Skinner ME Uzilov AV Stein LD Mungall CJ Holmes IH JBrowse a next generation genome browser Genome Res 2009 19 1630 1638 Keseler IM Bonavides Martinez C Collado Vides J Gama Castro S Gunsalus RP Johnson DA Krummenacker M et al EcoCyc a comprehensive view of Escherichia coli biology Nucleic Acids Res 2009 37 D464 D470 Byrne KP Wolfe KH The yeast gene order browser combining curated homology and syntenic context reveals gene fate in polyploid species Genome Res 2005 15 1456 1461 Dehal PS Joachimiak MP Price MN Bates JT Baumohl JK Chivian D Friedland GD et al
3. We have attempted to make JContex tExplorer easy to install and use by offering our program as a GUI WebStart application launching is as simple as navigating to a website and clicking on the appropriate button Additionally our program is organized in a way that does not require a steep learning curve among pro spective users To help new users we provide an extensive user manual and a series of video tutorials Additional file 1 along with the program executable Additional file 4 We hope that JContextExplorer may find use in the bio informatics community with its emphases of producing a positive user experience and simultaneously offering a nav igable tool of high quality and portability Availability and requirements Project Name JContextExplorer Operating System Platform independent Programming language Java Other requirements None License Source code and binary executable are available under terms of the GPL free software license ver sion 2 or later at http www bme ucdavis edu facciotti Seitzer et al BMC Bioinformatics 2013 14 18 Page 6 of 8 http www biomedcentral com 1471 2105 14 18 9 00 8 00 7 00 6 00 5 00 4 00 3 00 2 00 1 00 0 00 Acetobacter_pasteurianus_IFO_3283 01 1 Acetobacter_pasteurianus_IFO_3283 01 3 Dickeya_dadantii_3937 1 Dickeya_zeae_Ech1591 2 Erwinia_billingiae_Eb661 1 Erwinia_pyrifoliae_Ep196 2 Erwinia_pyrifoliae_Ep196 4 Gluconacetobacter_diazotrophicus_PAIS 1 Gluconacet
4. for eukaryotic genomes Genome Res 2003 13 2178 2189 36 Chen F Mackey AJ Stoeckert CJ Roos DS OrthoMCL DB querying a comprehensive multi species collection of ortholog groups Nucleic Acids Res 2006 34 D363 D368 37 Pandit S Gupta S A COMPARATIVE STUDY ON DISTANCE MEASURING IEEE Trans Neural Netw 2011 2 29 31 38 Pope SD Chen L L Stewart V Purine utilization by Klebsiella oxytoca M5al genes for ring oxidizing and opening enzymes J Bacteriol 2009 191 1006 1017 39 Liebert MA Masip L Veeravalli K Georgiou G The many faces of glutathione in bacteria Antioxid Redox Signal 2006 8 753 763 doi 10 1186 1471 2105 14 18 Cite this article as Seitzer et al JContextExplorer a tree based approach to facilitate cross species genomic context comparison BMC Bioinformatics 2013 14 18 Submit your next manuscript to BioMed Central and take full advantage of e Convenient online submission e Thorough peer review e No space constraints or color figure charges e Immediate publication on acceptance e Inclusion in PubMed CAS Scopus and Google Scholar e Research which is freely available for redistribution Submit your manuscript at O BioMed Central www biomedcentral com submit
5. hpxW in an operon with at least 3 other genes Therefore by taking into account context as well as homology it is possible to accurately separate ggt genes from hpxW genes We used JContextExplorer to attempt to separate ggt genes from hpxW genes in 22 alpha and gamma proteo bacterial species based on differences between ggt and hpxW contexts We found that ggt and hpxW grouped into two major out branches Figure 3 Interestingly we discovered a third group where manual investigation revealed that it was unclear if these genes were ggt or hpxW data not shown Visualization of the contexts in the hpxW group revealed agreement with previously described hpxWXYZ structures and a comparison of a whole genome phylogenetic tree with the ggt hpxW con text tree Additional file 2 revealed good agreement among closely related organisms Details relating to the methods associated with the above analyses are also avail able Additional file 3 This investigation highlights the utility of combining automation generating the ggt hpxW context tree with manual interrogation investigation using the multi genome browser context viewer tool Conclusion Comparing genomic contexts across organisms is an ef fective but underutilized technique While a handful of custom approaches have been developed no universal plat form for cross species genomic context analyses has yet been produced We have developed JContextExplorer to address this need
6. list of author information is available at the end of the article C BioMed Central transcription units 3 4 which ultimately result in differ ences among gene regulatory networks 5 Genomic con text may also be helpful in elucidating details of horizontal gene transfer and duplication events 6 8 and has been used to improve upon sequence based gene annotation algorithms 9 11 and aid in the construction of protein protein association networks 12 In each of these investi gations a new method was created to meaningfully define and compare genomic contexts The existence of a fast ac curate user friendly context comparison tool could have aided these investigations and could encourage future researchers to incorporate genomic context analyses into their investigations In plant and animal species a number of tools interro gating synteny the degree that genes remain on corre sponding chromosomes and collinearity the degree that 2013 Seitzer 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 Seitzer et al BMC Bioinformatics 2013 14 18 http www biomedcentral com 1471 2105 14 18 genes remain on corresponding chromosomes and in order 13 have been developed
7. program with Seitzer et al BMC Bioinformatics 2013 14 18 http www biomedcentral com 1471 2105 14 18 a set of 22 annotated microbial genomes computationally predict operons in all organisms using an intergenic distance threshold of 20 nucleotides and load pre computed hom ology cluster information for 81 102 annotated genes on a 2 x 2 8 GHz Quad Core Intel Xeon processor with 16 GB of RAM and total memory of 2 TB Results JContextExplorer software usage JContextExplorer may be launched via downloadable exe cutable JAR file or directly through the Internet via Java Page 3 of 8 WebStart at http www bme ucdavis edu facciotti resour ces_data software The program is organized as a series of major and minor windows laid out in a semi hierarchical manner Figure 1 An initial welcome window invites the user to 1 specify the genomic working set the set of gen omes to investigate see Figure 2 and 2 include cross species homologous gene cluster information Individual annotated genomes should be formatted as tab delimited GFF files version 2 This information is imported into JContextExplorer by selecting either a directory containing a set of GFF files or an additional tab delimited mapping 7 Main Window Context Viewer Window Start Window a i ror ry _ L z aen ee r m mme Annotations Add Remove Color Legend Gene Cont
8. such as MCScanX 14 and i ADHoRe 15 The Ensembl project 16 also utilizes syntenic data These tools have many useful features how ever lack a powerful visualization methods Additionally they do not focus on microbial species In general genomic context comparison methods applied to microbial species 3 11 have been highly customized non GUI based and not readily extendable to other investigations However a number of rudimentary GUI platforms for exploration of annotated microbial genomes have been developed such as the Integrated Microbial Genome system IMG 17 which has developed a system that allows clickable naviga tion of one or more genomes 18 While the tool offers several alternative homology based clustering methods it does not have much flexibility in other aspects for ex ample genes may only be organized into groups called chromosomal cassettes according to a hard coded 300 bp intergenic distance threshold and there is no way to ex port graphical representations of genome contexts Several tools have focused on visualization of syntenic and collinear regions such as the plant genome duplication database 12 PLAZA 19 and Genomicus 20 These tools are most appropriate when investigating plant and animal species however and could benefit from additional user flexibility and control in their visualization and inter rogation of genomic segments A number of genome nav igable interfaces have be
9. 9 96 2896 2901 Itoh T Takemoto K Mori H Gojobori T Evolutionary instability of operon structures disclosed by sequence comparisons of complete microbial genomes Mol Biol Evol 1999 16 332 346 Wolf YI Rogozin IB Kondrashov AS Koonin EV Genome alignment evolution of prokaryotic genome organization and prediction of gene function using genomic context Genome Res 2001 11 356 372 Jensen LJ Kuhn M Stark M Chaffron S Creevey C Muller J Doerks T et al STRING 8 a global view on proteins and their functional interactions in 630 organisms Nucleic Acids Res 2009 37 0412 D416 Tang H Bowers JE Wang X Ming R Alam M Paterson AH Synteny and collinearity in plant genomes Science New York N Y 2008 320 486 488 Wang Y Tang H Debarry JD Tan X Li J Wang X Lee T h et al MCScanXx a toolkit for detection and evolutionary analysis of gene synteny and collinearity Nucleic Acids Res 2012 40 e49 Proost S Fostier J De Witte D Dhoedt B Demeester P Van de Peer Y Vandepoele K i ADHoRe 3 0 fast and sensitive detection of genomic homology in extremely large data sets Nucleic Acids Res 2012 40 e11 Flicek P Arnode MR Barrell D Beal K Brent S Carvalho Silva D Clapham P et al Ensembl 2012 Nucleic Acids Res 2012 40 D84 D90 Markowitz VM Korzeniewski F Palaniappan K Szeto E Werner G Padki A Zhao X et al The integrated microbial genomes IMG system Nucleic Acids Res 2006 34 0344 D348 Mavromatis K Chu K
10. MicrobesOnline an integrated portal for comparative and functional genomics Nucleic Acids Res 2010 38 0396 D400 Gomez S Fernandez A Montiel J Torres D Solving Non uniqueness in agglomerative hierarchical clustering using multidendrograms J Classif 2008 65 43 65 Seitzer et al BMC Bioinformatics 2013 14 18 Page 8 of 8 http www biomedcentral com 1471 2105 14 18 28 Lathe WC lll Snel B Bork P Gene context conservation of a higher order than operons Mol Biol 2000 13 25388 25392 29 Sharma AK Walsh Da Bapteste E Rodriguez Valera F Ford Doolittle W Papke RT Evolution of rhodopsin ion pumps in haloarchaea BMC Evol Biol 2007 7 79 30 Techtmann SM Lebedinsky AV Colman AS Sokolova TG Woyke T Goodwin L Robb F Evidence for horizontal gene transfer of anaerobic carbon monoxide dehydrogenases Front Microbiol 2012 3 132 31 Holland RCG Down TA Pocock M Prli A Huen D James K Foisy S et al BioJava an open source framework for bioinformatics Bioinformatics Oxford England 2008 24 2096 2097 32 Mutton P Arnaud B Java EPS graphics 2D http jlibeps sourceforge net 33 Altschul S Gish W Miller W Myers E Basic local alignment search tool J Mol Biol 1990 215 403 410 34 Enright AJ Van Dongen S Ouzounis C An efficient algorithm for large scale detection of protein families Nucleic Acids Res 2002 30 1575 1584 35 LiL Stoeckert CJ Roos DS OrthoMCL identification of ortholog groups
11. Seitzer et al BMC Bioinformatics 2013 14 18 http www biomedcentral com 1471 2105 14 18 BMC Bioinformatics SOFTWARE Open Access JContextExplorer a tree based approach to facilitate cross species genomic context comparison Phillip Seitzer Tu Anh Huynh and Marc T Facciotti Abstract Background Cross species comparisons of gene neighborhoods also called genomic contexts in microbes may provide insight into determining functionally related or co regulated sets of genes suggest annotations of previously un annotated genes and help to identify horizontal gene transfer events across microbial species Existing tools to investigate genomic contexts however lack features for dynamically comparing and exploring genomic regions from multiple species As DNA sequencing technologies improve and the number of whole sequenced microbial genomes increases a user friendly genome context comparison platform designed for use by a broad range of users promises to satisfy a growing need in the biological community Results Here we present JContextExplorer a tool that organizes genomic contexts into branching diagrams We implement several alternative context comparison and tree rendering algorithms and allow for easy transitioning between different clustering algorithms To facilitate genomic context analysis our tool implements GUI features such as text search filtering point and click interrogation of individual contexts and genomi
12. alpha and gamma proteobacterial species were constructed using BLAST 33 and tribe MCL 34 All ggt and hpxW genes naturally grouped into the same homology cluster Using JContextExplorer we defined a context set which we named D75 that placed all genes on the same strand within 75 nucleotides of each other into common gene groupings We constructed a context tree of the ggt hpxW homology cluster using the Common Genes Dice dissimilarity metric and Joint Between Within linkage function above The data segmented into two branches one of which corresponded to the previously described hpxW context green box and the other into a combination of the ggt context blue box and an undetermined third group Manual inspection of individual contexts in the third group might reveal that some members of this group belong with the ggt group and some with the hpxW group Additionally some members in this third unknown group could represent transitional cases between the hpxW and ggt gene a gene that performs the functions of both hpxW and ggt for example JContextExplorer s context viewer tool proved helpful in manually interrogating this third group Seitzer et al BMC Bioinformatics 2013 14 18 http www biomedcentral com 1471 2105 14 18 resources_data software Incorporation into commercial software under non GPL terms is possible by obtaining a custom license from the University of California URL http www b
13. c visualization via a multi genome browser We demonstrate a use case of our tool by attempting to resolve annotation ambiguities between two highly homologous yet functionally distinct genes in a set of 22 alpha and gamma proteobacteria Conclusions JContextExplorer should enable a broad range of users to analyze and explore genomic contexts The program has been tested on Windows Mac and Linux operating systems and is implemented both as an executable JAR file and java WebStart Program executables source code and documentation is available at http www bme ucdavis edu facciotti resources_data software Keywords Genomic context Genomic neighborhood Comparative genomics Java GUI Background As genomic sequencing becomes increasingly accurate cheaper and widespread the need for tools to meaningfully interpret whole organism genomic sequence data has increased While a large collection of tools are devoted to sequence homology and phylogenetic analyses 1 2 far less attention has been paid to tools designed to meaningfully compare gene neighborhoods or genomic contexts across species Differences among genomic contexts across spe cies may indicate changes in the organization of functional Correspondence mtfacciotti ucdavis edu Department of Biomedical Engineering One Shields Ave University of California Davis CA 95616 USA Genome Center One Shields Ave University of California Davis CA 95616 USA Full
14. e 2 of 8 tree are assembled into a multidendrogram using variable group agglomerative hierarchical clustering Previous gen omic context investigations often determined the genomic contexts of interest in a set of species and compared the observable differences in genomic contexts to a phylogen etic tree of the organisms 28 30 However genomic con texts do not always differ in ways that match species phylogeny especially when a number of horizontal gene transfer events have taken place 30 Our context tree ap proach offers an alternative to whole species or even single gene phylogenetic trees that emphasizes the arrangement size and spacing of individual genetic elements within a contextual region of DNA instead of nucleotide specific differences in the DNA The genomic contexts used to assemble context trees may be interrogated in an intuitive context viewer win dow and information associated with individual genes may be retrieved by button clicks Our software facilitates easy modification of parameters and enables interrogation of several alternative genomic contexts of interest simultan eously A balance of automation and manual control is essential for any software tool we have attempted to auto mate only essential processes such as tree computation and tree rendering and leave a great deal of control to the user Our motivation was to develop a novel general purpose genomic context comparison platform to both 1 ge
15. en developed such as the UCSC genome browser 21 the Gaggle genome browser 22 and JBrowse 23 Many genome browsers have been developed with a focus of interrogating one or a few model organism s of interest such as EcoCyc interrogating Escherichia coli 24 and the Yeast Gene Order Browser interrogating various species of yeast 25 While these tools are sophisticated in their visualization schemes they are limited in the species available for cross species com parisons MicrobesOnline 26 has developed a domain browser tool which allows one to analyze the domain content of homologous proteins across microbial species However this tool compares the domain content of one gene at a time rather than the organization of groups of genes and so is not appropriate for studying changes in genomic context A tool with broad applicability powerful multi genome visualization tools and a high degree of user control could complement the existing set of synteny and genomic con text comparison tools well To bridge the gap in genomic context comparison and visualization software we have developed a new tool JContextExplorer Our tool extends the Java Multidendrograms package 27 which allows for flexible computation re analysis and export of multiden drograms We apply the multidendrogram approach to a set of user supplied annotated genomes to create context trees genomic contexts which form the leaves of the Pag
16. ext Sets Information ADS Context Viewer Cluster 4 Erwinia_billingiae b661 2 at paneo oaase Laseno ma Erwinia_pyrifoliae_Ep196 1 1000 11090 16000 Pamoea_ananatis_LMG 20103 1 004000 003000 3000 Klebsielia_oxytoca_MSal 1 000 t0000 000 0000 Klebsiella paeumonise NTUH K2044 1 1676000 1681000 k si 91009 Kiebsielia_variicola_At 22 1 Osan M size F Cluster 10 show Coordinates i Show Surrounding Before 1000 nt After 1000 m O Stop O Type F Annotation Strand Normalize C Color Surrounding Update Contexts Figure 1 Layout of JContextExplorer windows A JContextExplorer operates via usage of an initial data loading frame followed by coordination of a main window and multi genome browser context viewer window top These frames have several associated child windows second row from the main window a scrollable list of gene annotations Annotations and a window to facilitate multiple loading and switching between alternative context sets Add Remove Context Sets from the context viewer window an alphabetized gene color legend Color Legend and pop up window of information relating to a specific gene Gene Information An enlarged view of the context viewer window B reveals a scrollable viewing area where genes are rendered as colors rectangles appearing above or below a centerline depending on their strandedness Genes are colored according to homology or associated annotation The Gene Information panel lower left hand corner
17. f YI Rogozin IB Grishin NV Koonin EV Genome trees and the tree of life Trends in genetics TIG 2002 18 472 479 Kuzniar A van Ham RCHJ Pongor S Leunissen JaM The quest for orthologs finding the corresponding gene across genomes Trends in genetics TIG 2008 24 539 551 20 21 22 23 24 25 26 27 Page 7 of 8 Price MN Huang KH Arkin AP Alm EJ Operon formation is driven by co regulation and not by horizontal gene transfer Genome Res 2005 15 809 819 Price MN Arkin AP Alm EJ The life cycle of operons PLoS Genet 2006 2 e96 Novichkov PS Rodionov DA Stavrovskaya ED Novichkova ES Kazakov AE Gelfand MS Arkin AP et al RegPredict an integrated system for regulon inference in prokaryotes by comparative genomics approach Nucleic Acids Res 2010 38 W299 W307 Zu M Esteban CD Deutscher J Pe G Horizontal gene transfer in the molecular evolution of mannose PTS transporters Mol Biol Evol 2005 22 1673 1685 Rogozin IB Makarova KS Murvai J Czabarka E Wolf YI Tatusov RL Szekely L Koonin EV Connected gene neighborhoods in prokaryotic genomes Nucleic Acids Res 2002 30 2212 2223 Kojima KK Kanehisa M Systematic survey for novel types of prokaryotic retroelements based on gene neighborhood and protein architecture Mol Biol Evol 2008 25 1395 1404 Overbeek R Fonstein M D Souza M Pusch GD Maltsev N The use of gene clusters to infer functional coupling Proc Natl Acad Sci USA 199
18. gous gene groupings across many species or multiple genomic regions within a single species rather than ex plore the genome of a single species Individual genes are rendered as colored rectangles oriented above or below a centerline to represent their placement on the forward above centerline or reverse below centerline strand Each segment is centered about the center of each gene grouping Below all rendered contexts a genomic display sub panel contains check box options to 1 show hide genomic coordinates 2 normalize displayed contexts according to strand which may allow for easier visual in spection of analogous contexts 3 display genes sur rounding each context that are not a part of the context and 4 color the genes surrounding the context if this is unchecked surrounding genes are displayed as gray Genes are colored according to homology or common an notation depending on the method used to generate the context tree Left clicking on individual genes within a rendered context brings up a pop up window displaying biological information related to each gene this informa tion may be modified in a gene information sub panel Right clicking enables exporting rendered contexts as an image and offers the option to display a gene color legend Middle clicking selects the clicked gene as well as all hom ologous genes or genes that with the same annotation depending on the initial search type displayed in
19. in the user manual Linkage methods and display options available in the ori ginal multidendrograms package 27 are re implemented here which allows for easy re computation of the context tree All generated trees appear as individual internal frames the user may therefore work on several alternative contexts at once changes in tree computation and render ing will affect only the tree in focus Individual leaves on the tree which each represent a single context set group ing are named by concatenating the name of the organism from which they derive to a serial number of the instance that a query match was found within that organism Indi vidual leaves on the tree may be selected by clicking on their name clicking the select all button or entering a leaf name search filter in the genomic context viewer tool search bar located below the tree Subsequent mouse clicks may bring up child windows either for 1 annota tions of the query matches for selected or 2 a multi genome browser window context viewer window As depicted in Figure 1A the start window main window and context viewer window are the central components of Seitzer et al BMC Bioinformatics 2013 14 18 http www biomedcentral com 1471 2105 14 18 the tool and various child windows are available within the main window and context viewer windows The context viewer window Figure 1B is a multi genome browser specifically designed to interrogate analo
20. ing the system locations of all individual annotated genomes files and corresponding species names The user may also include tab delimited cross species gene cluster ing information which could be computed using a com bination of BLAST 33 and MCL 34 for example or one of a number of various other gene clustering pipelines 35 36 Homology cluster information may be entered in 5 alternative tab delimited file formats please see the user manual for a more detailed description Once these files have been loaded the user pushes a submit button to close the starting window and open the main window Once in the main window of the system the user may search all loaded genomes by 1 gene annotation or 2 common homology group ID number All computed genomic groupings in all organisms that contain one or more genes that match the search query are retrieved and organized in to a multidendrogram according to a dissimilarity measure and linkage function As a default the starting context set defines genomic groupings only as the annotated features that match a search query called the SingleGene context set however 6 additional con text sets are available and may be accessed by clicking the Add Remove button in the starting frame Available gen omic grouping schemes include organizing genes into operons taking a range of nucleotides or genes around a query match and loading a customized set of genomic groupings from fi
21. le for a complete description please see the user manual In this program we have implemented 4 genomic grouping comparison metrics or dissimilarity measures each of which are appropriate for different use cases If the genomic groupings that comprise a given con text set are large we suggest using either Common Genes Dice or Common Genes Jaccard metrics which im plement the set based Dice and Jaccard dissimilarity approaches 37 with the individual annotated features within each grouping acting as elements and the whole genomic grouping acting as the set If genomic groupings contain the same annotated features however vary in the intergenic spacing between features we recommend using the Moving Distances approach which uses gene order and intergenic spacing to describe differences between contexts Changes in intergenic spacing between genes within an operon has been experimentally shown to be related to gene co expression in E coli and B subilits 4 and may be a reflection of microbial gene regulatory net works changing over evolutionary time 3 4 Finally if the context set under investigation does not appear to change significantly except in the size of one or more genes the Total Length dissimilarity metric may be effective this is especially useful in for genomic groupings that consist of only one or a few genes A more detailed description of these dissimilarity metrics is available
22. me ucdavis edu facciotti resources_ data software Additional files Additional file 1 ContextExplorer User Manual Comprehensive user manual including installation use instructions examples diagrams contact information and links to website tutorial videos source code and other resources Additional file 2 Supplementary Figures and Methods related to separating hpxW from ggt in 22 alpha and gamma proteobacteria Visualization of hpxW contexts comparison of whole species phylogeny to hpxW sub portion of JContextExplorer generated context tree and detailed description of methods associated with hpxW ggt analysis Additional file 3 Alpha and Gamma proteobacteria biological information Annotated genomes of 22 alpha and gamma proteobacterial species whole species phylogenetic tree for 22 alpha and gamma proteobacterial species and MCL tribe determined homology clusters for 22 alpha and gamma proteobacterial species Additional file 4 JContextExplorer version 1 07 Executable JAR file of the latest version of the JContextExplorer program Program may be launched on any computer with the Java runtime environment JRE installed The program may be launched either from a command line or by double clicking on the program icon Competing interests The authors declare they have no competing interests Authors contributions PS wrote the source code and drafted the manuscript PS and TH analyzed the hpxWXYZ ope
23. nerate context trees and 2 facilitate genomic explor ation through our multi genome browser interface We demonstrate a use case for our tool by resolving annotation ambiguities between ggt and hpxW genes among 22 species of alpha and gamma proteobacteria Though in the use case provided here we focus on microbial species we emphasize that analyses are not limited to microbial species Implementation JContextExplorer is a platform independent pure Java ap plication requiring Java 1 6 or higher The software extends the MultiDendrograms software package 27 and also uses BioJava 31 and the Java EPS Graphics2D API version 0 1 32 The software has been tested to func tionally equally on MacOS X Windows 7 and Linux Ubuntu environments Input data is read in via a series of tab delimited text files We provide instructions and exam ples in the user manual Additional file 1 to help fam iliarize new users to the tool The look and feel of all GUI components has been set to match the default look and feel of the operating system running the program Program development was undertaken over several platforms to en sure an intuitive look and feel on all major platforms JContextExplorer has the ability to output JPG PNG and EPS representations of context tress and multi genome browsable contexts EPS representations of genomic contexts were achieved using the Java EPS Graphics2D API 32 It took approximately 35 seconds to launch the
24. obacter_diazotrophicus_PAIS 3 Gluconobacter_oxydans_621H 1 Klebsiella_pneumoniae_NTUH K2044 1 Klebsiella_variicola_At 22 1 Marinomonas_sp_MWYL1 2 Pantoea_ananatis_LMG_20103 2 Pantoea_vagans_C9 1 1 Pantoea_vagans_C9 1 2 Pectobacterium_wasabiae_WPP163 2 Serratia_proteamaculans_568 1 Serratia_proteamaculans_S68 2 Teredinibacter_turerae_T7901 1 Teredinibacter_turnerae_T7901 2 Xanthomonas_campestris_ATCC_33913 3 Yersinia_enterocolitica_subsp_palearctica_Y11 1 Acetobacter_pasteurianus_IFO_3283 01 2 Gluconobacter_oxydans_621H 2 Erwinia_pyrifollae_Ep196 3 Gluconobacter_oxydans_621H 3 Parvularcula_bermudensis_HTCC_2503 2 Xanthomonas_campestris_ATCC_33913 1 Xanthomonas_campestris_ATCC_33913 2 Gluconobacter_oxydans_621H 4 Acidithiobacillus_ferrooxidans_ATCC_53993 1 Erwinia billingiae_Eb661 2 Cellvibrio_japonicus_Ueda107 1 h xW Marinomonas_sp_MWYL1 1 p Tolumonas_auensis_OSM_9187 1 Teredinibacter_turnerae_T7901 3 Gluconacetobacter_diazotrophicus_PAIS 2 Parvularcula_bermudensis_HTCC_2503 1 Dickeya_dadantii_3937 2 Dickeya_zeae_Ech1591 1 Erwinia_billingiae_Eb661 3 Klebsiella_oxytoca_MSal 1 Klebsiella_pneumoniae_NTUH K2044 2 Klebsiella_variicola_At 22 2 Pantoea_ananatis_LMG_20103 1 Pantoea_vagans_C9 1 3 Pectobacterium_wasabiae_WPP163 1 Serratia_proteamaculans_568 3 Yersinia_enterocolitica_subsp_palearctica_ 11 2 Erwinia_pyrifoliae_Ep196 1 Figure 3 hpxW and ggt context tree Clusters of all homologous gene clusters in 22
25. ron in alpha and gamma proteobasterial species with TH which was instrumental in the development of JContextExplorer TH also helped write the background information regarding purine catabolism and the hpxW gene in alpha and gamma proteobacteria in the supplemental information MF provided essential feedback for software development and oversaw the project and helped to interpret results related to the hpxWXYZ genomic contexts All authors contributed to the preparation of the manuscript and have read and approved the final manuscript Acknowledgements Erin Lynch provided instrumental feedback for the development of the program especially from the point of view of essential biologically meaningful features Aaron Darling provided helpful advice for the Java implementation and aid in running the PhyloSift program Dr Valley Stewart provided expert knowledge of the hpxWXYZ operon in alpha and gamma proteobacterial species and aided in discussions of the project Support for PS and MTF came from NSF EF 094953 and startup funds to MTF Author details Department of Biomedical Engineering One Shields Ave University of California Davis CA 95616 USA 7Genome Center One Shields Ave University of California Davis CA 95616 USA 3Microbiology Graduate Group One Shields Ave University of California Davis CA 95616 USA Received 10 October 2012 Accepted 21 December 2012 Published 16 January 2013 References l Wol
26. the frame Finally the rendered range of each context may be easily changed using the range around context segment sub panel and clicking an update contexts button The context viewer window and main window are actively linked modifying selected leaves in the tree for example will add or remove these leaves in the context viewer win dow after clicking the update contexts button The tool is designed to facilitate coordination of the context tree and the context viewer window such coordination may inspire re investigation of the same gene of interest using alternative context groupings or re computation of the context tree using a different clustering algorithm Analysis of the hpxW and ggt genes in 22 alpha and gamma proteobacteria In the gamma proteobacterial species Klebsiella oxytoca MSal the hpxW gene is known to form an operon with hpxW hpxY and hpxZ 38 The hpxW gene however is highly homologous to another gene encoding gamma glutamyl transpeptidase ggt A sequence alignment of K oxytoca hpxW and Escherichia coli ggt revealed that their amino acid sequences are almost co linear and share 30 identity This high degree of homology con fuses automated annotation programs which often Page 5 of 8 misannotate hpxW as get Fortunately the ggt enzyme has been characterized in several microbial organisms 39 and has a genomic context very different from the hpxW context ggt occurs as a single gene
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