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1. in the database overlapping the GLADxX retrieved ortholog sequence to be considered as the same prediction The default value is 70 G Activation of the gladx driver agent to automate the search of lineage specific losses on all nodes Activation of this agent allows to analyze systematically the lineage specific losses from all nodes available along the lineage leading to the input reference from the selected ancestor Targets 9606 is a parameter defining the species concerned by lineage specific loss searched by GLADX It allows to focus the search on the interest species When no species are specified GLADX searches all lineage specific losses along the studied lineage By default the value is empty The activation of agents are defined with these following markups lt master gt lt type gt Agent_Name lt type gt lt master gt By default gladx_driver agent is deactivated by comment markups To activate it the comment markups of the gladx_driver agent must be removing and the line of the orthologs_group_mode parameter of the geneloss_event_search agent must be commented Note When new studies are performed with the gladx_driver agent its orthologs_group_mode lineage TaxidAncestor parameter is used to define from which ancestor the study begin While if the gladx_driver agent is launched after a first round of analysis with default mode its orthologs_group_mode lineage TaxidAncestor parameter does not us2. it exists a rake in NCBI we have noted it by In these case we have decided one topology and chose a name for the ancestors When an ancestor name does not exist due to a topology incoherent between our choice and the NCBI topology there is noted Taxid identifiers For each leaves and ancestral nodes it exists an unique taxonomic identifier These identifiers are numbers that are noted close to leaves or ancestral nodes To follow our topology two ancestor names were invented and consequently no taxid exist so we have tied to the ancestral species a negative unique identifier On these 22 species by default 21 species of Euteleostomi are studied because the orthologs_group_mode parameter defined in the home tower TOWER_1 03 prod DGH_2 dagobah xml file is parameterized to analyze losses in Euteleostomi taxid 117571 in lineage mode However the analysis of largest phylum such as Chordates including Ciona is conceivable by using the taxid 7711 In the contrary smallest phylum could be studied by using the taxid of any ancestor described in the figure 1 The number of species studied in a phylum may be modified by choosing among the 22 species those kept in the scope parameters species_scope_for_phylogeny_study amp species_scope_list_for_phylogeny_study Produced data and results Results are automatically produced as report files and databases contents Report files can be easily read by our user friendly viewer FG
3. tree topology 2 1 Database modifications 2 2 Advise the length of branches Can I use an other kind of protein database GLADX parameters Introduction GLADxX is a module included in a software application DAGOBAH Gouret et al 2011 According to its name Gene Loss Analyzer DAGOBAH eXtension it is dedicated to gene losses and pseudogenizations automatic detection and analysis DAGOBAH relies on other relevant software tools FIGENIX Gouret et al 2005 PhyloPattern Gouret et al 2009 IODA In press http ioda univ provence fr All these components form the lab s bioinformatic software platform called T O W E R Tools Operating With Evolutive Resources GLADX work in the TOWER framework For us and for external users TOWER is now very complex to install because one has to deploy many software components many bioinformatics binaries many databases and many genomic data So we chose the virtualization strategy that means the installation of all TOWER s components on a virtual machine image Several image instances can be started as virtual computers on computers which disposes of a virtualisation software like VirtualBox VMWare and on Clouds Technical requirements We decided to build an Ubuntu 11 04 64 bit image on VirtualBox 4 1 2 Oracle TM Therefore this image will work efficiently on 64 bit architecture host computers To run one image of TOWER we recommend using a four cores workstatio
4. Gene Loss Analyzer DAGOBAH eXtension GLADX User s Manual EBM Evolutionary Biology and Modeling Laboratory UMR 7353 Aix Marseille University France Download the VirtualBox GLADX image 17Go Link to benchmark analysis of 14 reported cases Currently only available for identified users The current GLADX version enables using 22 species from Ensembl v57 Bos taurus Canis familiaris Ciona intestinalis Danio rerio Equus caballus Gasterosteus aculeatus Gallus Gallus Gorilla gorilla Homo Sapien Monodelphis Domestica Meleagris Gallopavo Macaca Mulatta Mus Musculus Ornithorhynchus anatinus Oryzias latipes Pan Troglodytes Pongo Pygmaeus Abelii Rattus norvegicus Sus scrofa Taeniopygia guttata Tetraodon nigroviridis Xenopus Tropicalis GLADX corresponds to a set of agents The sources are freely available and could be retrieved in the home tower TOWER_1 03 prod DGH_2 src directory The distributed GLADX version could easily be modified as described in the procedure below in order to increase the number of studied species Thus GLADX can also function with any species present in Ensembl from the version 48 to 58 included giving the option to work with 51 different species Table of contents Introduction Technical requirements GLADX launch Choice of phylum and species studied Produced data and results How to add new species retrieved from Ensembl 1 Install proteome and or genome 2 Create the
5. PathOfTheSpeciesDNAFile The proteomes of species already present are in the file nome tower TOWER _ 1 03 prod FGX_API GenomicDB ensembl To add new species you need to add the proteome in this file and re formatted it command formatdb in Blast package Note When you add a new proteomes or and a new genomes you need to format the FASTA headers as follows gt Icl ENSP00000375415 9606 HOMO SAPIENS corresponding to gt Icl SequenceName Taxid Species Name OptionalyADescription 2 Modify the tree topology The binary species tree defined in GLADX needs to contain the species chosen for analyses 2 1 Database modifications The tree topology of species is provided into FIGENIX database called figenix_db in the dagobahtreeoflife table The topology is described branch by branch where each taxid is linked to its parent taxid and a description of its rank class if it is an ancestral node species if it is a leaf An ancestral node must be linked to two taxid corresponding to their child nodes Warning if you add new species that are outgroup of species already present the farthest ancestor must always be linked to the ghost root taxid 1 2 2 Advise the length of branches The length of branches of the species tree topology is defined in the file home tower TOWER_1 03 prod DGH_2 src project_specific pl as follows tof_branch_length_to_node taxid branch_length You need to add all the new branch length
6. XView home TOWER FGX View The most important result is the final species tree of species set in which all the results are pinpointed Databases contents are of two kinds FIGENIX results produced on a SGBDR PostgreSQL in the database figenix_db DAGOBAH results produced as an ontological database see supplement 1 that relies also on a SGBDR PostgreSQL database dagobah_db Note that these databases can be deployed on our IODA web site through collaborations 1 Manipulate databases figenix_db and dagobah_db in SQL To manipulate these databases please use the following commands in a Terminal e To backup the database in SQL format pg_dump DatabaseName f SavingFileName e To delete a database dropdb DatabaseName e To create a database createdb DatabaseName e To install a new database psql DatabaseName f DatabaseTolInstall Note DatabaseTolInstall may be a database saved earlier or a clean figenix_db or dagobah_db database available in the directory home tower Examples Databases Warning There is an incompatibility with the SGBDR PostgreSQL when the version gt 8 2 is used we used 8 4 When a new database is created before database installation you must be connected to the database as postgres user sudo su postgres then psql DatabaseName and past the text present in the home tower jena_with_postgres_higher_than_8 2 file 2 Manipulate ontological database only dagobah_db in OWL The ontological re
7. ed In this case all the nodes of the lineage are analyzed from the ancestor that was defined at first round in the orthologs_group_mode lineage TaxidAncestor of geneloss_event_search agent
8. er to continue the study at nucleotide level The default value is 50 jdentity_threshold 50 is the minimum identity threshold needed between an orthologous sequence retrieved by GLADX and a known protein to continue the study at nucleotide level The default value is 50 jdentity_threshold_for_real_gene 70 is the minimum identity threshold needed between known protein and used reference protein to be used in study at nucleotide level The default value is 70 E Parameters defined in the genelosses_synthetic_analysis agent nucleotide_in_more_by_side 10000 is the number of nucleotides taken on each side of an orthologous gene to build an alignment with orthologs retrieved during the study It is the step just before the reconstruction The value must be identical to the geneloss_event_search value The default value is 10000 F Parameters defined in the verify prediction existence agent When GLADX retrieves an ortholog it systematically checks the database used to see whether there is an annotation on its position Sometimes previously described genes are present on the same area overlap_threshold 50 is the minimum overlap threshold in percentage for a previously described gene in the database to consider that they are on the same position The default value is 50 jdentity_threshold_to_conclude_gene_already_exist 70 is the minimum identity threshold in percentage for a previously described gene
9. n with 4Go of RAM minimal configuration Our image is configured as a default to run with eight cores and with 8 Go current workstation producing our tests Please note that hardware virtualization technology has to be activated on the host computers VT X for Intel AMD V for AMD in order to obtain most advantageous performances Warning the hyperthreading technology with OpenMPI that is a software layer used to exploit parallel computing with bioinformatics softwares like Tree Puzzle ClustalW is not recommended because of reported bugs Note the eight cores are not strictly required for the image users could modify the scripts as below e in home tower TOWER_1 03 prod FGX_API scripts puzzle_cmd_perl change np 8 by np X where X is the number of cores you want to use e same procedure for home tower TOWER_1 03 prod FGX_API scripts clustalw_cmd e same procedure for home tower TOWER_1 03 prod FGX_API pipelines Templates __CassiopePhylo M __ replace a 8 in block lt nodeRef gt blast lt nodeRef gt lt parameterAssignation gt lt parameterName gt options lt parameterName gt lt parameter Value gt a 8 lt parameterValue gt About the network configuration of the image the NAT mode was set as a default This mode doesn t allow ssh access but it is very much faster than Bridge mode Images can be run with or without X Window GUI quite slow in the emulation In NAT mode RDP clients ca
10. n be used to access to a non graphical image In Bridge mode one can use ssh Important the tower user has tOwer as password in ssh or graphical mode GLADX image is downloadable on the following link GLADX image First uncompress it then add it to VirtualBox with the GUI or with vboxmanage registervm command GLADX launch GLADX is started on boot of the image with VirtualBox In order to start a gene study with GLADxX one just has to deposit one or several FASTA files amino acid sequences in the following directory home tower GLADX_DATA The FASTA files require to be named as follows EnsembIProteinSequenceName Taxid fasta This file must contain a sequence in FASTA format with an header in the following format gt Icl EnsembIProteinSequenceName Taxid Species Name OptionalyADescription corresponding in this actual example gt Icl ENSP00000375415 9606 HOMO SAPIENS A golden dataset of 14 FASTA files corresponding to the cases reported at http ioda univ provence fr is available in the directory home tower Examples Fastas Additional options Users can deactivate automatic start of GLADX on boot of the image by commenting with a the line su tower c home tower TOWER_1 03 prod DGH_2 start of the file etc rc local In this configuration you need to launch DAGOBAH using the command start in a Terminal from the current directory home tower TOWER_1 03 prod DGH_2 To stop DAGOBAH you just need to pres
11. quences present in this subtree form an orthologous group From this orthologous group it deduces the lineage specific losses comparing the species present in the group to the species set selected for the study An agent allowing to analyze systematically all nodes of the lineage leading to the input reference from the selected ancestor can be activated gt see G section In species mode it searches in the phylogeny the species that have orthologs to the reference protein given as input until the TaxidAncestor ancestor and deduces losses comparing species that have an ortholog to the species set selected for the study The default value is lineage 117571 that corresponds to a search of species that have no representative of a gene established at least since the last common ancestor of Euteleostomi do_not_study_when_species_exist 9606 9544 defines species that will stop the study if an ortholog exists in the first phylogeny Should be empty if you want to analyse all the species where the gene is missing If you need to concentrate on losses in a specific species note its taxid here If a database described ortholog already exists for your species in the first phylogeny there is no need to continue the study to save your time By default the value is empty minimum_size_of_orthologs_group_for_begin_the_study 3 is the minimum size of an ortholog group required in the first phylogeny to continue the study The defaul
12. s Note When you change the version of Ensembl data you must change the value of the maxEnsemblBuildNumber parameter with the corresponding Ensembl version This parameter is in the file home tower TOWER_1 03 prod DGH_2 ENSJHelper properties Particular Case If you add new species that are outgroup of chordate you need to change the taxid of the far ancestor of the new tree topology in the home tower TOWER_1 03 prod DGH_2 src project_specific pl file replaced the taxid defined in dagobah_treeoflife_database_root 7711 Can I use an other kind of protein database Yes but only in simple mode To use the simple mode you need to modify the dagobah xml file available at this path home tower TOWER_1 03 prod DGH_2 1 change the mode as described below search_missing_cause_in_genome no 2 change the path to your new database database Path_database_used GLADX parameters Numerous parameters are available to adjust the behaviour of GLADX Some are essential such as species and used database ortholog detection mode from the used reference sequence or from its ortholog the most exterior depending to the selected phylum and mode of study verification of putative lost genes or not These parameters must be defined before analysis is launched They are contained in an XML file accessible at home tower TOWER_1 03 prod DGH_2 dagobah xml The parameters of agents are defined between the following markups l
13. s CTRL C or alternatively to kill the process Choice of phylum and species studied The default parameters of GLADX allow to analyze lineage specific gene losses in Euteleostomi or from the closest ancestor in leaves direction whether the gene is appeared later by studying the orthologous group containing the protein reference given as input By default 22 chordates species are used with the topology described below Tree of life of 22 species implemented in GLADX 3 Homo sapiens 06 207598 Pan troglodytes 9598 a Gorilla Gorilla A Pongo abelii 96 Macaca mulatta 44 95 Canis familiaris Equus caballus 9796 Sus scrofa 9823 56 m ol 791561 32525 Wena Bos taurus Rattus norvegicus 10116 Mus musculus 10090 Monodelphis domestica 13616 Ornithorhynchus anatinus 32523 ee aenio la guttata 59729 pygia g Gallus gallus 9031 Meleagris gallopavo 9103 117571 lt RAN Xenopus tropicalis 64 Tetraodon nigroviridis 99883 Gasterosteus aculeatus 69293 Oryzias latipes 90 ryz p 7711 80 Danio rerio 7955 Ciona intestinalis 19 Date of speciations They come from Time Tree http www timetree org and are displayed by million of years my When the date was not available it is indicates by and we have chose a coherent date Species and ancestor names The names are taken from NCBI taxonomy When a common ancestor remain unclear and
14. sults can be exploited by Protege software and exported in OWL files A script named clear_with_file is available at the directory home tower TOWER_1 03 prod DGH_2 It allows to delete a database and to install a new database from an OWL file In this case you need to be in the DGH_2 directory and use the command in a Terminal like this clear_with_file dagobah_model CompletePathOfTheDatabaseName owl Example to install a dagobah_db ontological database empty clear_with_file dagobah_model home tower Examples Databases dagobah_db_empty owl To backup the dagobah_db database in owl without use of Protege you need to be in the directory DGH_2 and launch the following command owldump NameOfBackup owl How to add new species retrieved from Ensembl The current GLADX version enables using 22 species but more species can be used by some manipulation 1 Install proteome and or genome Genomes are required when you use GLADX in complete mode parameter search_missing_cause_in_genome in the dagobah xml file The genomes of species already present are in the directory home tower TOWER_1 03 prod FGX_API GenomicDB ensembl_dna To add new species you need to add the formatted command formatdb in Blast package genome in this directory You need also to add the path of the formatted file containing the DNA in the file home tower TOWER_1 03 prod DGH_2 src project_specific pl like this species_dna_database Taxid
15. t engine def gt lt type gt Agent_Name lt type gt lt engine def gt A Parameters defined in the fasta protein phylo agent species_scope_for_phylogeny_study 9598 9606 9544 10116 10090 9601 9615 8090 9031 13616 7719 8364 99883 9593 9103 9913 9796 9823 9258 59729 69293 7955 and species_scope_list_for_phylogeny_study 9598 9606 9544 10116 10090 9601 96 15 8090 9031 13616 7719 8364 99883 9593 9103 9913 9796 9823 9258 59 729 69293 7955 are two identical species scopes identified by taxid with different formats employed to choose species used during the study Phylogenies will be built with these species The default value is that described above 22 species database Path_database_used defines the path of the protein database used The default path is AlgoTools Blast db ensembl B Parameters defined in the geneloss_ event search agent nucleotide_in_more_by_side 10000 is the number of nucleotides taken on each side of a TBLASTN hit to output a prediction value must be identical to the genelosses_synthetic_analysis value The default value is 10000 orthologs_group_mode mode TaxidAncestor is the ortholog sequence analysis mode launched There are two mode options lineage or species cf article In lineage mode GLADX searches the sub tree having the TaxidAncestor ancestor as root and containing the reference given as input All the se
16. t value is 3 search_missing_cause_in_genome choice defined if you want to use GLADX in complete mode to search for the genome of a species where orthologs are missing in the first phylogeny Choice can be yes or no If no is chosen no verification of loss is made and the results output come exclusively from analyses of the first phylogeny built from the chosen database making the process much faster The default value is yes translate_in_gene_to_detect_ortholog_if_necessary choice is defined when you have a tree of proteins that you want to translate into genes Allows comparing two ortholog groups of a gene or two ortholog groups of a protein Choice can be yes or no No is faster but a little less precise force_to_analyse_this_species 9593 9606 This parameter allow to annotate the list of selected species even if an ortholog is found by phylogeny in the first step By default the value is empty C Parameters defined in the best hit fgx agent max_nb_managed_hits 5 is the number of hits retained from TBLASTN to continue the analysis More this number is high more the GLADX analysis can be long when putative tested homologous sequences are not orthologous The default value is 5 D Parameters defined in the genelosses checkpoint all events by study agent length_threshold 50 is the minimum overlapping threshold between an orthologous sequence retrieved by GLADX and a known protein in ord
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