***MAKER DOCUMENTATION*** ALSO SEE THE GMOD MAKER TUTORIAL http://gmod.org/wiki/MAKER_Tutorial #--------------------------------------------------------------------- INSTALLING MAKER !!INSTALLATION INSTRUCTIONS FOR MAKER ARE IN .../maker/INSTALL!! Following installation the maker exectutable will be in .../maker/bin. If .../maker/bin doesn't exist then you haven't finished installing. The following directories will be found in the MAKER installation (some are generated during setup): bin - contains the MAKER executables. lib - contains necessary perl libaries for MAKER. src - installation folder for configuring and installing MAKER. exe - MAKER installed external execuatables are stored here. perl - MAKER compiled and localized CPAN libs are stored here. data - contains sample data for use by MAKER. GMOD - contains files to assist in configuring other GMOD tools (See sections titled APOLLO and JBrowse below) MWAS - contains optional MAKER web based GUI Now you can run MAKER!! Just type maker -h on the command line to see the usage statement Programs required by MAKER rely on certain environmental variables being set. If you have not set these variables per the installation instructions of the external programs, a reminder list is provided below: for tcsh: setenv ZOE where_snap_is_installed setenv AUGUSTUS_CONFIG_PATH where_augustus_is_installed/config for bash: export ZOE=where_snap_is_installed export AUGUSTUS_CONFIG_PATH=where_augustus_is_installed/config Note: ZOE should not be set to .../snap/Zoe, rather just .../snap #--------------------------------------------------------------------- MAKER WITH MPI !!INSTALLATION INSTRUCTIONS FOR MPI ARE IN .../maker/INSTALL!! To run with MPI, run MAKER via mpiexec. Example: (This will run MAKER on 4 nodes or processors) mpiexec -n 4 maker maker_opts.ctl maker_bopts.ctl maker_exe.ctl Please see the documentation of the MPI environment you use for instructions on how to initiate an MPI process. #--------------------------------------------------------------------- HOW TO USE MAKER Step-by-step GMOD tutorial --> http://gmod.org/wiki/MAKER_Tutorial MAKER mailing list --> http://box290.bluehost.com/mailman/listinfo/maker-devel_yandell-lab.org Search List archives --> https://groups.google.com/forum/#!forum/maker-devel *Please search the archives before sending questions to the list #--------------------------------------------------------------------- RUNNING MAKER WITH EXAMPLE DATA 1) Copy the files in the data directories to a temporary directory where you will run an example file. 2) Type maker -CTL to generate generic MAKER control files 3) Next you will need to edit the control files to include the path of the genome file, EST file, and protein file, as well as the paths to all required executables. See CONFIG FILE EDITING for more information. 4) Then try the following command from your temporary directory: maker maker_exe.ctl maker_opts.ctl maker_bopts.ctl 5) Examine the output files. See MAKER OUTPUT and APOLLO sections. #--------------------------------------------------------------------- CONTROL FILE EDITING MAKER uses control files to guide each run. Generic control files can be built using the -CTL flag in maker. These control files can then be edited by the user to identify the location of all required input data and statistics. Control files are run specific and separate control files will need to be built for each genome given to MAKER. MAKER will look for control files in the current working directory, so it is recomended that MAKER should be ran in a separate directory containing unique control files for each genome. Control files: 1. maker_exe.ctl - contains the path information for needed executables. 2. maker_bopts - contains filtering statistics for BLAST and Exonerate. 3. maker_opts.ctl - contains all other information for MAKER, including the location of the input genome file. Remember to examine the control files before each run of MAKER on your specific data. Lines in the MAKER control files have the format key=value with no spaces before or after the equals(=). If the value is a file name, you can use relative paths and environmental variables, i.e. genome=$HOME/my_genome.fasta For most options that take a file name, you can supply multiple files using comma seperated lists. i.e. est=dmel.fasta,cele.fasta You can also add optional labels to the output using a colon(:) to seperate a file name from the label. Labels will be passed on the the output GFF3. This is convenient for seperating out tiers for GBrowse or JBrowse. i.e. est=dmel.fasta:fly,cele.fasta:nematode Note that for all control files the comments written to help users begin with a pound sign(#). In addition, options before the equals(=) can not be changed, nor should there be a space before or after the equals. A. maker_exe.ctl - includes information about programs executed by MAKER. Here is an example of section (not everything) from the maker_opts.ctl file: #-----Genome genome=/fastas/genome.fasta #-----EST Evidence est=/fastas/est.fasta altest=/fastas/alt_est.fasta #-----Protein Homology Evidence protein=protein.fasta #-----MAKER Specific Options evaluate=0 max_dna_len=100000 min_contig=1 min_protein=0 split_hit=10000 pred_flank=200 single_exon=0 single_length=250 keep_preds=0 map_forward=0 retry=1 clean_try=0 clean_up=0 #--------------------------------------------------------------------- MAKER OUTPUT MAKER will create at least the following files/directories: 1) XXX.maker.output/ - contains all output for a given run of MAKER. 2) XXX.maker.output/XXX_datastore/ - contains subdirectories that hold the output for each individual contig of the input fasta file. See DATASTORE DIRECTORY STRUCTURE section. 3) XXX.maker.output/XXX_master_datastore_index.log - log of MAKER run progress as well as an index for traversing through the output datastructure. 4) XXX.maker.output/mpi_blastdb/ - Contains fasta indexes and error corrected fasta files built from the EST and protein databases provided by the user. 5) maker_opt.log,maker_exe.log,maker_bopts.log - These are logs of the control files used for this run of MAKER. 5) XXX.maker.output/XXX.db - Database of GFF3 files provided by the user. See GFF3 PASSTHROUGH section. Within the XXX_datastore/ subdirectories: * seq_name.gff - a gff file that can be loaded into GMOD, GBROWSE, or Apollo * seq_name.maker.transcripts.fasta - a fasta file of the MAKER annotated transcript sequences * seq_name.maker.proteins.fasta - a fasta file of the MAKER annotated protein sequences * seq_name.maker.XXX.transcript.fasta - a fasta file of ab-initio predicted transcript sequences from program XXX * seq_name.maker.XXX.proteins.fasta - a fasta file of ab-inito predicted protein sequences from program XXX * seq_name.maker.non_overlapping_ab_initio.transcripts.fasta - a fasta file of filtered ab-inito transcript sequences that don't overlap maker annotations * seq_name.maker.non_overlapping_ab_initio.proteins.fasta - a fasta file of filtered ab-inito protein sequences that don't overlap maker annotations * theVoid.seq_name/ - a directory containing all of the raw output files produced by MAKER, including BLAST reports, SNAP output, exonnerate output and the masked genomeic sequence. IMPORTANT: * The names of output files are based on sequence ids. If giving MAKER a multi-fasta file, it is important to verify that all sequence id are unique, so files are not overwritten. * Output is store din a deep datastore structure. See DATASTORE DIRECTORY STRUCTURE in this document. * If sequence IDs contain characters that are illegal in file names, those characters will be replaced automatically before building output file names. #--------------------------------------------------------------------- DATASTORE DIRECTORY STRUCTURE Many filesystems have performance problems with large numbers of subdirectories and files within a single directory, and even when the underlying filesystems handle things gracefully, access via network filesystems can be an issue. You can imagine that the amount of output produced while annotating an entire genome can be quite overwhelming to the file system. To deal with all the output files MAKER uses a Datastore module to create a hiearchy of subdirectory layers, starting from a 'base', and mapping identifiers to corresponding subdirectory. A deep datastore will be used by MAKER if there are more than 1,000 sequences in a multi-fasta file. When a deep datastore is implemented, MAKER output files will not appear where you would normally expect them to be. Instead they will be located in a series of sub-directory under a new base-directory whose name is determined based on the input genome file name: EXAMPLE: current_directory/fly_datastore/EE/Af/Contig1/Contig1.gff To help you locate output files, a master_datastore_index file is created which lists the exact output directory corresponding to each contig from the input genome file. The The master_datastore_index file contains three columns of text; the first column shows the sequence identifier from each fasta header, and the second column shows the location of the output files for that sequence. The third column is for logging the status of data related to an individual contig. The values of the third column are as follows: * STARTED - Indicates that MAKER has started proccessing this contig. * FINISHED - Indicates that MAKER has finished processing this contig and all data is currently available in that subdirectory. * DIED - Indicates that MAKER failed on this contig. * DIED_SKIPPED_PERMANENT - Indicates that MAKER failed up to the specified number of retries and will not try again. * RETRY - Indicates that MAKER is retrying the contig after a failure. * SKIPPED_SMALL - Indicates that this contig was skipped because it is too short (based on control file values set by the user). #--------------------------------------------------------------------- GFF3 PASSTHROUGH If you have data from a source that MAKER does not support, and you wish to use the data in annotating a genome, then you can pass the data to MAKER as an aligned GFF3 file. This is done by supplying the files location to the appropriate value in the maker_opt.ctl file (i.e. est_gff=some_file.gff). Note that MAKER expects all data sent to it to be of the type specified, so don't put mixed data in a file (i.e. don't mix EST and other data in the file pointed to by est_gff, otherwise it all gets used as EST data). Also the maker_gff option is only for MAKER produced GFF3 files. Other GFF3 files of mixed data must be split by type and identified by the appropriate control file option (i.e. rm_gff for repeat data, pred_gff for ab-initio prediction data, est_gff for EST data, etc.). You should use the online GFF3 validator to see if your GFF3 files comply with all GFF3 specifications before running MAKER: http://modencode.oicr.on.ca/cgi-bin/validate_gff3_online #--------------------------------------------------------------------- APOLLO MAKER is bundled with a configuration file that improves the color and display of MAKER annotations and evidence in the Apollo genome browser. The configuration file is called "gff3.tiers" and is located in the maker/GMOD/Apollo/ directory. The file should be copied to the ~/.apollo sub_directory in your home directory. You may have to create ths directory if it doesn't exist. You will need to use a terminal window to do this. Example: mkdir ~/.apollo cp maker/GMOD/Apollo/gff3.tiers ~/.apollo #--------------------------------------------------------------------- CHADO MAKER is bundled with a script to make it easy to load MAKER results into a chado database. The script is maker2chado. You will also want to review CHADO documentation at http://www.gmod.org/. You can also use the chado2gff3 script bndled with MAKER to dump from Chado to GFF3. #--------------------------------------------------------------------- JBROWSE MAKER is bundled with a configuration file that improves the color and display of MAKER annotations in JBrowse. The is configuration file is maker.css. Place it in your JBrowse directory. You can then use the script maker2jbrowse to automatically load MAKER produced GFF3 into JBrowse. #--------------------------------------------------------------------- HMM BUILDING (for training SNAP, based on snap documentation) A) First you will need to determine the genes used to model future genes, by determining a high quality gene set (annotations for the high quality gene should be in GFF3 format). The high quality gene set can then be coverted into snap ZFF format using maker2zff.pl found in maker/bin. This program is run with the following command: maker2zff.pl <directory> genome * <directory> a the directory where all of your GFF3 files are located * geneome is the name for the outfile Files Created: genome.ann genome.dna Note: A convenient way to identify and initial high quality gene set for the HMM is to use the -predictor est2genome option in MAKER. This will produce gene annotations based solely on EST evidence. These annoations can then seed the first HMM. After running MAKER again using this new HMM and the -predictor snap option, you can use the second round of annotations as the seed for an even better HMM model. In this way the HMM model progressively improves with each run of MAKER. Another strategy for identifying an initial gene set to model the HMM is to use the program CEGMA (http://korflab.ucdavis.edu/ software.html). CEGMA builds a highly reliable set of gene annotations in the absence of experimental data by identifying DNA regions with homology to a set of 458 proteins that are highly conserved among taxa. Use cegma2zff.pl to convert cegma output for training SNAP. Combining both CEGMA and MAKER datasets to build the first HMM is also a good strategy. B) Next you will use the dna and zff file (genome.dna and genome.ann) to produce a SNAP HMM as described in the SNAP documention (which we have provided below): The first step is to look at some features of the genes: fathom genome.ann genome.dna -gene-stats Next, you want to verify that the genes have no obvious errors: fathom genome.ann genome.dna -validate You may find some errors and warnings. Check these out in some kind of genome browser and remove those that are real errors. Next, break up the sequences into fragments with one gene per sequence with the following command: fathom -genome.ann genome.dna -categorize 1000 There will be up to 1000 bp on either side of the genes. You will find several new files. alt.ann, alt.dna (genes with alternative splicing) err.ann, err.dna (genes that have errors) olp.ann, olp.dna (genes that overlap other genes) wrn.ann, wrn.dna (genes with warnings) uni.ann, uni.dna (single gene per sequence) Convert the uni genes to plus stranded with the command: fathom uni.ann uni.dna -export 1000 -plus You will find 4 new files: export.aa proteins corresponding to each gene export.ann gene structure on the plus strand export.dna DNA of the plus strand export.tx transcripts for each gene The parameter estimation program, forge, creates a lot of files. You probably want to create a directory to keep things tidy before you execute the program. mkdir params cd params forge ../export.ann ../export.dna cd .. Last is to build an HMM. hmm-assembler.pl my-genome params > my-genome.hmm Lastly, you will want to add the location of your hmm file to your maker_opts.ctl file. * For more information see SNAP documentation on how to build an HMM