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1. Current RF Score 309 00 Current RF Score 307 00 Curre 6 Baikal_omul goby mosquito_fish guppy medeka_fish Sphoeroides pigeon anole tiger_salamander Figure 3 5 Displaying the output species tree 10 Chapter 4 Available Features This chapter gives a description of available features in MulRF s user interface The menu bar contains four item File Analysis Options and Help 4 1 File The File menu item assists in selecting the input files to run MulRF analyses Figure 4 1 Its drop down menu contains following items eoe MulRF Analysis Options Help Open Gene Tree File G Open Scoring File AS Exit 3 Q Figure 4 1 File menu item in MulRF 4 1 1 Open Gene Tree File This allows the user to select an input gene tree file for the species tree search from folder inputData in the project home directory The menu item is disabled as soon as file is selected and it remains disabled until the input file is closed The same command can also be executed by typing CTRL G 4 1 2 Close Gene Tree File This enables the user to close a previously selected gene tree file Closing a gene tree file enables the user to select another input gene tree file using the Open Gene Tree File 11 4 1 3 Open Scoring File This menu item allows the user to select a scoring file from the folder inputData in the project home directory The menu item is disabled as soon as a file is selected and it rem2. to gene tree discord MulRF implements fast local search based heuristics which greatly speed up species tree inference 1 2 It provides the following features e The input gene trees can be weighted for the species tree inference e Topological constraints can be imposed on the inferred species tree e Running multiple replicates of the tree search heuristic is automated e Weighted or un weighted MulRF distances of a given species tree from a collection of input trees as well as the MulRF distance to each gene tree can be computed e The input gene trees and the resulting species tree can be illustrated NOTE Throughout this manual we describe the input trees as multi copy gene trees However MulRF is not limited to gene tree input Indeed it can be used to build a supertree from any collection of single and or multi labeled trees including gene trees species trees or taxonomy trees Chapter 2 Using MulRF This chapter gives an overview of how to use MulRF Topics include downloading steps file formats and user interfaces 2 1 Download MulRF is an open source program and the source code is available for download at https github com ruchiherself MulRFRepo If you do not want to compile the source code the platform independent binary executable is available at http genome cs iastate edu CBL MulRF This executable has been successfully tested on Windows 7 Mac OS X 10 9 and Ubuntu 12 04 To install the b
3. 23 1 00 Tree 3 33 1 00 Tree 4 33 1 00 Tree 5 57 1 00 Tree 6 32 1 00 Tree 7 8 1 00 Tree 8 40 1 00 Tree 9 20 1 00 Tree 10 3 1 00 Figure 3 2 After selecting the input gene tree file 2 Now click Analysis gt Tree search This step is typically the most time consuming depending on the input size and details of the heuristic search Observe the log window for continuous updates Figure 3 3 3 Once the tree search is over a statistics table for the output folder appears The rows in this table correspond to the generated output files inside the new folder under outputData folder Figure 3 4 The first column in the output folder statistics table has buttons to display the statistics table of the corresponding files The second column shows the MulRF score of the resulting species tree in each file Clicking the file name button shows the statistics table of the file Figure 3 4 It lists species tree followed by input gene trees with their score weight and View buttons to display them Observe that the weight of the first gene tree is 0 thus this gene tree does not have any impact on the species tree search 000 MulRF File Analysis Options Help o Y Log MULA CIL A JLUL TC JUL LU Current RF Score 377 00 Current RF Score 373 00 Current RF Score 369 00 Current RF Score 367 00 Current RF Score 365 00 Current RF Score 363 00 Current RF Score 361 00 Current RF Score 359 00 Current RF Score 357 00 Current
4. MulkRF A platform independent software package for phylogenetic analysis using multi copy gene trees Version 1 2 User s Manual Contents Introduction Using MulRF 25 Download MI A Se RE A RS Bt BRE eng ds ta 2 2 Input Output File Format aaa aa 22 1 Geneand Speci s Tree Files sc ba Ge he tk Be Be eee Se 2 22 Constraints Flert d os rt Ne eo BO at BR o Oh A aa Be d 22 3 Scoring Fue dw prats arta er aa Dedede a 2 24 Output File wg 4 6 cig a A A AA AA A es 2 3 User Interface 4 he esas sue baleen d shy deb gd Se Se we ated aise he Bales ZII LOS WINDOWS 6 ii Mee Ba OE BS es A ee he A POS Oe See ee 2 3 2 Tree Display WindOWS sa oce i dis ec a Ae ee ee A 233 Table WindOWS i mara ies BAA oe eR ad a BOM I Ae a Sample Run Available Features ACL El fn dd Sees ie ae en A Sa ho Be ce nye ee ted 4 11 Open Gene Tree Files a Docs Sb A bea SO Ga DA ee Vea 4 1 2 Close Gene Tree File ee 413 Open Scoring Files sansi oS ta E Ga Ee ne e les he Gt ys ee le ote 41 4 Close Scoring Fil uri a a e ee a ae ee a a Be a Bd Aico SE XAG Ae hele Be eas Cece Pt PAS AR te cat Bade ed NS Ot RR IS ee ts AS Pade 4 2 GATNALY SIS patito ala a ate dh A E une generation ae wh le AA wee di AD Mi APECSEATCH tu nd Sova toss eke fe BO theives ect do Ml thet Shao Cte WP heh Bledsoe ty Hoa 422 MUIRE Score sees cord ite guia PA A e Goatees a pha es wi AS Options et F s an Bat A E POA oe els ay Cade Bee BOLE MAS O
5. RF Score 355 00 000 W vertebrates02 newick Stats Name Leaves Weight View Tree Tree 1 73 Tree 10 Figure 3 3 Running tree search The generated output files are now available inside a new folder under outputData Observe the name of the newly generated folder in the log window The output species tree or gene trees can be displayed by clicking the View button in the output file statistics table Figure 3 5 The contents of the log window are stored inside logFile under the project home directory o Y Log CUCU NE OCUIC JA UU Current RF Score 313 00 Current RF Score 311 00 Current RF Score 309 00 Current RF Score 307 00 Current RF Score 305 00 Current RF Score 303 00 Current RF Score 303 00 Displaying the Statistics Table of the generate la Displaying the Statistics Table of File vertebri Y vertebrates02 newick Stats Name Leaves Weight View Tree 73 E View Tree 10 Figure 3 4 Statistics tables for the newly generated output folder and file Output Folder Stats File Name MuIRF Score vertebrates020ut1 303 00 Gene Tree1 Gene Tree2 Gene Tree3 Gene Tree4 Gene Tree5 Gene Tree6 Gene Tree7 Gene Tree8 Gene Tree9 Gene Tree10 2 0 File Analysis Options Help 8000 Y Log OO O W Output Folder Stats _ n File Name MuR O O O vertebrates02Out1 newick Stats vertebrates020ut1 i Current RF Score 311 00
6. Seed gt Figure 4 3 Options menu item in MulRF 4 3 1 Starting Species Tree Generation This allows the user to choose the method to be used to obtain the initial starting species tree for the heuristic search There are two available choices 1 Leaf Adding This is a greedy step wise method to build an initial starting species tree it is the default choice This method considers the input trees as rooted by using the default Newick format tree roots and uses the rooted MulRF distance The rooted MulRF distance is computed based on the the rooted RF distance instead of unrooted RF distance between the extended species tree and the multi copy gene tree The greedy step wise method randomly selects three leaves from the species leaf set and considers all the triplets that these three leaves can form It randomly selects a triplet with the lowest rooted MulRF score based on the input gene trees as the backbone tree Next it randomly selects a new leaf species to add to the initial 3 taxon backbone tree The algorithm tries to add this leaf in all possible positions in the current tree and it stores all those positions that give the best score lowest rooted MulRF distance to the input gene trees It then randomly selects a position from among these best locations and adds the leaf at that location This process of adding a new leaf is repeated until the starting tree includes all species Once all the leaves are added we have our initial spec
7. ains disabled until the scoring file is closed The same command can also be executed by typing CTRL S 4 1 4 Close Scoring File This menu item closes the previously selected scoring file After closing the scoring file the user can use Open Scoring File to select another scoring file 4 1 5 Exit Clicking the exit submenu item closes quits the application This can also be done by typing CTRL Q in Windows and Command key Q in Mac OS 4 2 Analysis This menu item assists in initiating the heuristic tree search and computing the MulRF score Figure 4 2 Its drop down menu contains following items 4 2 1 Tree search This submenu allows to start the heuristic search Note that the gene tree file should be opened and options if required should be set before clicking it and beginning the tree search 4 2 2 MulRF score This submenu allows to compute the MulRF distance from a given species tree to a collection of gene trees eoo MulRF Fie EUAS Options Help Tree search MulRF score Figure 4 2 Analysis menu item in MulRF 12 4 3 Options This menu item assists in customizing the heuristic tree search Figure 4 3 These options should be set if required before initiating the heuristic species tree search The drop down menu contains following items eoo MulRF File Analysis Options Help Starting species Tree Generation Output Folder Name No of Replicates gt Constraints File Random
8. ator rabbit cow pig mouse rat human eel trout chum_salmon atlantic_salmon chum_salmon Baikal_omul carp catfish sea_bass tilipia tilipia lungfish Japanese_toad rabbit horse cat pig whale cow sheep goat macaque human hamster mouse rat turtle turkey chicken alligator alligator crocodile crocodile gecko anole chicken cave_fish goldfish cave_fish cave_fish goat human marmoset human chicken pigeon goldfish Xenopus chicken human cow mouse rat chicken cave_fish goldfish gecko anole chicken cave_fish goldfish goldfish Japanese_lamprey lamprey anole skate eel cave_fish zebrafish goldfish carp Baikal_omul goby medeka_fish mosquito_fish guppy tiger_salamander Xenopus bull_frog frog alligator chicken dog cow sheep hamster mouse rat human rabbit tunicate plaice dogfish cod fugu atlantic_sblmon atlantic_salmon lamprey lamprey lamprey lamprey lamprey chicken human human human human mouse rat rat cow dog rat rat pig dog cow chicken chicken atlantic_salmon Xenopus Xenopus medeka_fish frog turtle chicken quail mouse human dog Pacific_hagfish inshore_hagfish fugu cherry_salmo
9. au J G Burleigh and D Fern ndez Baca Assessing approaches for inferring species trees from multi copy genes Under review 2 R Chaudhary J G Burleigh and D Fern ndez Baca Inferring species trees from incongruent multi copy gene trees using the robinson foulds distance Algorithms for Molecular Biology 28 8 2013 3 C M Zmasek and S R Eddy ATV display and manipulation of annotated phylogenetic trees Bioin formatics 17 383 384 2001 16
10. be specified by the prefix WEIGHT lt value gt If a weight is not specified the input tree is given a weight of 1 Here is an example of assigning weight to an input tree amp WEIGHT 0 5 speciesA speciesB speciesC WARNING Due to rounding errors fractional weights can sometimes cause MulRF to malfunction However weights like 0 5 or 0 25 work fine 2 2 2 Constraints File A constraints file allows the user to impose constraints on the topology of the inferred species tree As the name indicates the file contains a set of topological constraints for the species tree Each constraint is a comma separated list of species names terminated with a semicolon In the resulting species tree the species in a constraint will be closer to each other than to the rest of the species Multiple constraints can be specified but any single species can be part of at most one constraint For an example a constraint file may contain the following two constraints cat dog turtle rice arabidopsis NOTE The constraints option can only be used if the leaf adding option is selected to generate the initial species tree 2 2 3 Scoring File A scoring file is used to compute the MulRF distance from a given set of gene trees to a species tree A scoring file contains a species tree followed by a list of gene trees A scoring file has the same format as a gene tree and species tree file Section 2 2 1 except that the first tre
11. e in a scoring file must be a species tree 2 2 4 Output File The first tree in the output file of MulRF search is the species tree with lowest MulRF score found during the heuristic search The total reconciliation cost of this species tree appears in a comment right before this tree The species tree is followed by a list of all the input gene trees with the individual MulRF score of each gene tree against the species tree appearing in a comment with that tree All the input files should be stored inside the inputData folder under the project home directory After execution output files are stored in a unique folder inside the outputData folder under the project home directory The default output file folder name is a system generated hexadecimal number followed by the input file name 2 3 User Interface MulkRF s user interface includes a log window to record results of analysis gene and species tree display windows and various gene tree species tree and output folder table windows 2 3 1 Log window The log window displays all the selections made in the drop down menu options and updates the user on the species tree search in the real time It appears at any first action in MulRF s main window and can be found in the top left corner throughout the analyses The log window scrolls down to display the latest inserted line The contents of the log window are also saved to a text file logFile in the project home directory 2 3 2 T
12. ies tree Note that the root of this initial species tree will not be considered further in the heuristic search 2 User Specified This allows the user to choose a file containing a species tree in Newick format This user supplied species tree is then used as the starting species tree for the heuristic search 4 3 2 Output Folder Name This submenu item allows the user to specify a name for the folder in which the output files with the results of the analysis will be stored The default name for the output folder is composed of input gene tree file name followed by a unique system generated hexadecimal number 13 4 3 3 No of Replicates The tree search heuristic is a randomized algorithm and the search may get caught in a local minima To explore the tree space more thoroughly we recommend executing the tree search heuristic multiple times on the same data set MulRF automates this by running multiple replicates each using a different random seed to build the starting tree and to order the edges of the species tree for subtree prune and regraft SPR operations MulRF allows the user to enter the number of replicate runs to be performed The No of Replicates submenu provides the following two choices 1 1 Default This sets the number of replicates to the default value 1 2 Customize This opens an input dialog box where the user can change the default to any input number less than 100 4 3 4 Constraints File This menu ite
13. inary executable simply download and unzip MulRF1 2 zip or MulRF1 2 tar gz for Linux to a computer For Mac and Windows move inside the directory MulRF1 2 and click the executable jar file Mu1RF to begin For Linux and others open the terminal shell and move to the directory MulRF1 2 Then type java jar MulRF jar NOTE 1 MulRF requires Java Runtime Environment JRE 5 0 or later version to be installed on the computer to run 2 Linux users should set the execute permissions of executables in MulRF1 2 gt executables before running MulRF 2 2 Input Output File Format 2 2 1 Gene and Species Tree Files A gene tree file is an ASCII format text file containing a collection of input gene trees The gene trees must be in Newick format terminated by a semicolon and they can be non binary unresolved In the gene trees the leaf names must correspond to a species name e g the name of the species from which the gene was sequenced MulRF allows multi copy gene trees as input i e gene trees that have multiple leaves gene copies with the same label or name corresponding to a single species Here is an example of a multi copy gene tree in Newick format speciesA speciesB speciesC speciesA The following rules must be followed when creating a gene tree or species tree file The species names in the species tree must be unique occur only once in the tree Labels with non alphabetic characters e g
14. k has ten multi copy gene trees see Figure 3 1 This input gene tree file is located in inputData folder inside MulRF s home directory eoo A vertebrates02 newick amp WEIGHT 0 0 tunicate amphioxus inshore_hagfish Pacific_hagfish lamprey Japanese_lamprey skate dogfish Alopias electric_ray eel cod goby guppy mosquito_fish killifish medeka_fish plaice tilipia sea_bass sea_bream Sphoeroides fugu Baikal_omul trout cherry_salmon chum_salmon atlantic_salmon zebrafish white_sucker cave_fish catfish goldfish carp lungfish newt tiger_salamander salamander Japanese_toad Xenopus frog bull_frog Scleropus anole cobra gecko turtle crocodile alligator ostrich pigeon goose duck turkey quail chicken whale pig cow goat sheep rabbit marmoset macaque human horse guinea_pig hamster rat mouse cat fox dog lamprey lamprey atlantic_salmon cobra bull_frog Xenopus Xenopus chicken rabbit horse human dog pig sheep cow rat mouse rat horse human mouse rat rat 3 tunicate killifish killifish Xenopus Xenopus Xenopus Scleropus duck chicken pigeon pigeon turtle alligator mouse human pig killifish lamprey dogfish human fox mouse rat chicken Scleropus turtle allig
15. m allows the user to select constraints file to impose constraints on the topology of the inferred species tree Clicking of Constraints File submenu opens the file chooser window to select the constraints file from inputData folder under the project home directory 4 3 5 Random Seed The random seed allows the user to specify the seed for the random number generator used by MulRF There are two available choices 1 System Generated Default The default random seed is the system wall clock time 2 Customized This allows the user to enter their own random seed This is helpful when the user wishes to repeat a particular analysis Analyses using the same random seed the same data set and the same search options will produce the same output 4 4 Help This menu item provide help to the MulRF user Figure 4 4 Its drop down menu contains following two items 4 4 1 About This provides a brief information about MulRF 4 4 2 Help Contents This is designed to guide the user through the available features in MulRF s user interface The items in the left can be clicked to see the corresponding details in the right section of the help content window A search feature is also provided to search and see the details of different features The same command can also be executed by typing CTRL H 14 File Analysis Options La About Help Contents Figure 4 4 Help menu items in MulRF 15 Bibliography 1 R Chaudhary B Bouss
16. n white_sucker white_sucker white_sucker fugu chum_salmon chum_salmon cherry_salmon lamprey electric_ray lungfish Japanese_toad frog duck Japanese_toad ostrich lungfish horse human pig cow sheep sheep rat mouse quinea_pig whale pig cow sheep human human mouse rat ostrich chicken duck amphioxus Xenopus Pacific_hagfish medeka_fish Alopias mouse Scleropus goose turkey Xenopus Xenopus Pacific_hagfish Alopias mouse human Xenopus goose turkey newt salamander sea_bream sea_bream Sphoeroides Figure 3 1 Input gene tree file To estimate an optimal species tree for this collection of gene trees the user must launch MulRF and perform the following steps 1 Click File gt Open Gene Tree File anda file chooser window will appear Select vertebrates02 newick and click Open This will set a log window at the top left corner and a statistics table of the selected gene tree file The Input File Statistics Table lists gene trees with their number of leaves weights and View buttons Figure 3 2 The log window shows the selected input file name 000 MulRF File Analysis Options Help 00 0 YY Log DATE 2014 06 03 14 11 31 Jee SE Eee eee oe tate Selected Gene Tree File vertebrates02 newick y vertebratesO2 newick Stats Name Leaves Weight View Tree Tree 1 73 0 00 View Tree 2
17. ree Display Windows MuIRF displays gene and species tree by converting their Newick file into PNG format tree image using ATV 3 NOTE MulRF returns an unrooted species tree and the root in the tree display window is arbitrary 2 3 3 Table Windows e Input File Statistics Table Upon selection of an input gene tree file MulRF creates an input file statistics table It lists all the input gene trees with their leaf counts weights and view buttons to display them Only one gene tree file can be selected at a time e Output Folder Statistics Table After a species tree search MulRF creates an output folder statistics table It lists all the generated output files as a button with the MulRF distance of the species tree in each file from the collection of input gene trees e Output File Statistics Table Clicking the output file button in the output folder statistics table creates an output file statistics table It lists the inferred species trees followed by a list of input gene trees used in the analysis This table also includes the score weight and a view button for each tree The score of a species tree is its MulRF distance from all the input gene trees For an input gene tree the score denotes the MulRF distance of it from the species tree listed above it Chapter 3 Sample Run Here we present an example of how to run MulkRF to infer a species tree from a sample data set Our sample input file vertebrates02 newic
18. s eee 4 3 1 Starting Species Tree Generation oo 43 2 Output Fold r Names s s eva eS sa e a RE Me ee Ss 4 3 3 No of Replicates ima hoe GMA eA Re A e 4 34 Constraimts File 33 450 eo A A RE ee A WO oe a eS 4 3 5 Random Seed 2 uaa ea a a a a E a ee A A A E E E A E E EE E E E Bos 8 WNNNN 44 1 About 4 4 2 Help Contents ii List of Figures 3 1 3 2 3 3 3 4 3 5 4 1 4 2 4 3 4 4 Input genetresttile cdas Ma A io eo O mk a e e MU Ad e A A 6 After selecting the input gene tree file o o oooo ooo e e 7 Running trees archiiA posta a E ds ADS OER Aw EES Be Eee a 8 Statistics tables for the newly generated output folder and file 9 Displaying the output species tree ooo 10 File menu item in MulRF ee 11 Analysis menu item in MulRF o ooo 12 Options menu item in MulRF aoaaa aaa 13 Help menu items in MURE Genre genns e a BS a eee ee a es eg 15 iii Chapter 1 Introduction MulRF is a platform independent software package for inferring species trees from incongruent multi copy gene trees using a generalization of the Robinson Foulds RF distance to multi labeled trees i e trees with multiple leaves having the same label The underlying generic tree distance measure and fast running time makes MulRF useful for analyzing large genomic data sets in which multiple evolutionary processes as well as phylogenetic error may contribute
19. spaces must be encapsulated in apostrophes or quota tion marks e g speciesA species A or species A A comment in the input file must be encapsulated in a square brackets A comment can appear any where including inside the Newick format tree e g speciesA speciesB example comment text speciesC A gene tree can span multiple lines Gene trees can be unrooted or rooted which is specified by the prefix amp U or amp R for unrooted or rooted cases respectively A gene tree without any prefix is considered rooted If an input tree is rooted the root will not be considered during the species tree search instead the gene tree will be treated as an unrooted topology Also if an input gene tree has a root then this root may get lost in the tree search that means if the gene tree is rewritten in the output file then its default Newick root may differ from its initial root The input trees can have branch lengths but MulRF does not use branch length data to calculate the MulkF distance or infer the species tree If a gene tree is weighted the MulRF distance for that weighted gene tree is multiplied by its weight Thus gene trees with a higher weight should have a larger impact in the tree search The weight can be any positive real number from 0 to 1 A weight of 0 means that the gene tree has no effect on the MulRF analysis i e it is equivalent to not including the gene tree in the analy sis A weight can
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