root/README

***MAKER Documentation***

#----------------------------------------------------
INSTALLATION INSTUCTIONS FOR MAKER

*Step by step instructions are also available in the INSTALL text file.

MAKER is an annotation pipeline.  In other words it links together many steps and programs to produce final annotations.  For this reason, you must first install a number of programs that MAKER depends on.


To install maker, you will first need to install the following external programs:

     *PERL 5.8.0 or higher
     *BioPerl 1.5 or higher (www.bioperl.org)
     *SNAP version 2009-02-03  or higher (homepage.mac.com/iankorf)
     *RepeatMasker 3.1.6  or higher (www.repeatmasker.org)
     *Exonerate 1.4  or higher (www.ebi.ac.uk/~guy/exonerate)

You must also install one of the following:

     *Wu-BLAST 2.0  or higher (Wu-BLAST is becoming AB-BLAST which can not yet be downloaded)
        or
     *NCBI BLAST 2.2.X or higher (http://www.ncbi.nlm.nih.gov/BLAST/download.shtml)
 
You might want to also install these optional external programs:

     *Augustus 2.0  or higher (augustus.gobics.de)
     *GeneMark.hmm-E 3.9 or higher (exon.biology.gatech.edu)
     *FgenesH (www.softberry.com/) - requires licence

To install mpi_maker, you must have an mpi package installed, try the following:

     *MPICH2 (http://www.mcs.anl.gov/research/projects/mpich2/)

note:  Remember to install MPICH2 with the --enable-sharedlibs flag set to the appropriate value (See MPICH2 Installer's Guide at http://www.mcs.anl.gov/research/projects/mpich2/documentation/index.php?s=docs).


Notes:
1) Wu-BLAST is becoming AB-BLAST.  Once AB-BLAST becomes available we will do some testing to see if it is compatible with MAKER.  Wu-BLAST is no longer available online, so if you don't already have it, you will have to use NCBI BLAST instead.
2) RepeatMasker requires Wu-BLAST or Cross_Match and a single file executable called TRF (see RepeatMasker website for details), so please install these before installing RepeatMasker
3) Exonerate Binaries can be downloaded from the website.  If you use Mac OSX, however, binaries are only available for version 1.0.  This verion will work too.  If you would like to compile exonerate, it requires GLIB, a C-library, that has a link from the exonerate website.  If you use Mac OSX, GLIB can downloaded using FINK.
4) RepeatMasker requires a repeat library file, which can be downloaded from Repbase upon registration (http://www.girinst.org/), this is explained on the RepeatMasker website.
5) Please note the location of all of the programs that you have installed, and add them to you $PATH variable in your .profile file.  You will need this information in the maker.exe file, one of MAKER's 3 control files.


Now that you have all the necessary programs installed, MAKER can be unpacked using:

tar xvfz maker.tar.gz

This will create a directory called maker with 5 sub directories:

        bin - contains the maker executables.
        lib - contains all the necessary perl libaries for MAKER.
        MPI - contains MPI specific data to configure MAKER for a cluster that supports MPI.
        Apollo - contains gff3.tiers file (See section titled APOLLO below)
        data  - contains some sample data used to make sure everything works

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 seperate control 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 seperate 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.


Always remember to be examine the control files before each run of MAKER on your specific data


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 PERL5LIB where_bioperl_is_installed
setenv WUBLASTMAT where_wublast_is_installed/matrix
setenv ZOE where_snap_is_installed
setenv WUBLASTFILTER where_wublast_is_installed/filter
setenv AUGUSTUS_CONFIG_PATH where_augustus_is_installed/config

for bash:
export PERL5LIB=where_bioperl_is_installed
export WUBLASTMAT=where_wublast_is_installed/matrix
export ZOE=where_snap_is_installed
export WUBLASTFILTER=where_wublast_is_installed/filter
export AUGUSTUS_CONFIG_PATH=where_augustus_is_installed/config


#----------------------------------------------------
MPI MAKER INSTALL

If you are running maker on an MPI capable cluster, you can install an MPI version of maker by doing the following:

        1. Install standard maker and verify that it runs.
        2. Install MPICH2 with the --enable-sharedlibs flag set to the appropriate value (See MPICH2 documentation)
        3. Use cd to change to the MPI subdirectory in the maker instalation folder (i.e. maker/MPI/)
        4. Run Install.PL by typing:     perl Install.PL

A new version of maker called mpi_maker should now be installed under maker/bin.

To run mpi_maker, first verify that your mpi environment is initiated, (i.e. using the mpdboot or mpd command). Now start mpi_maker via mpiexec.

Example: (This will run MAKER on 3 nodes or processors)

        mpiexec -n 3 perl maker_directory/maker/bin/mpi_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.


#----------------------------------------------------
MAKER USAGE STATEMENT

Usage:

     maker [options] <maker_opts> <maker_bopts> <maker_exe> <evaluator>

     Maker is a program that produces gene annotations in GFF3 file format using
     evidence such as EST alignments and protein homology.  Maker can be used to
     produce gene annotations for new genomes as well as update annoations from
     existing genome databases.

     The four input arguments are user control files that specify how maker
     should behave. The evaluator options file contains control options specific
     for the evaluation of gene annotations. All options for maker should be set
     in the control files, but a few can also be set on the command line.
     Command line options provide a convenient machanism to override commonly
     altered control file values.

     Input files listed in the control options files must be in fasta format.
     Please see maker documentation to learn more about control file
     configuration.  Maker will automatically try and locate the user control
     files in the current working directory if these arguments are not supplied
     when initializing maker.

     It is important to note that maker does not try and recalculated data that
     it has already calculated.  For example, if you run an analysis twice on
     the same dataset file you will notice that maker does not rerun any of the
     blast analyses, but instead uses the blast analyses stored from the
     previous run.  To force maker to rerun all analyses, use the -f flag.


Options:

     -genome|g <filename> Specify the genome file.

     -predictor|p <type>  Selects the predictor(s) to use when building
                          annotations.  Use a ',' to seperate types (no spaces).
                          i.e. -predictor=snap,augustus,fgenesh

                          types: snap
                                 augustus
                                 fgenesh
                                 genemark
                                 est2genome (Uses EST's directly)
                                 abinit (ab-initio predictions)
                                 model_gff (Passes through GFF3 annotations)

     -RM_off|R           Turns all repeat masking off.

     -retry   <integer>  Rerun failed contigs up to the specified count.

     -cpus|c  <integer>  Tells how many cpus to use for BLAST analysis.

     -force|f            Forces maker to delete old files before running again.
                         This will require all blast analyses to be rerun.

     -evaluate|e         Run Evaluator on final annotations (under development).

     -quiet|q            Silences most of maker's status messages.

     -CTL                Generate empty control files in the current directory.

     -help|?             Prints this usage statement.


#----------------------------------------------------
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:

perl maker_directory/bin/maker maker_exe.ctl maker_opts.ctl maker_bopts.ctl

MAKER will create at least the following files/directories:

XXX.maker.output/ - contains all output for a given run of make
XXX.maker.output/XXX_master_datastore_index.log - log of MAKER run progress as well as an index for traversing XXX.maker.output/XXX_datastore/
XXX.maker.output/XXX_datastore/ - contains folders containing the output for each individual contig of the input fasta file
*Within these folders 
        seq_name.gff - a gff file that can be loaded into GMOD, GBROWSE, or Apollo
        seq_name.maker.transcripts.fasta - a file of the maker transcript sequences
        seq_name.maker.proteins.fasta - a file of the maker protein sequences
        seq_name.maker.XXX.transcript.fasta - a file of ab-inito transcript sequences from program XXX
        seq_name.maker.XXX.proteins.fasta - a file of ab-inito protein sequences from program XXX
        seq_name.maker.non_overlapping_ab_initio.transcripts.fasta - a file of filtered ab-inito transcript sequences that don't overlap annotations
        seq_name.maker.non_overlapping_ab_initio.proteins.fasta - a file of filtered ab-inito protein sequences that don't overlap 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 sequence

WARNING:
*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.
*If there are more than 1,000 sequences in a multi-fasta file a deep datastore structure will be used. see DATASTORE 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

"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.  The Datastore modules create a hiearchy of subdirectory layers, starting from a 'base', and mapping end-user's identifiers to the corresponding subdirectory." - quote from http://www.yandell-lab.org/  (See site for more information on the Datastore module)

A deep datastore will be used by maker if there are more than 1,000 sequences in a multi-fasta file.

When a datastore is implemented, the output files described above 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 from the input genome file name, i.e. current_working_directory/genome_datastore/EE/Af/Contig1/Contig1.gff.  A master_datastore_index file will be made in the current working directory to help you find the output files from each sequence.

The master_datastore_index file is a file created to allow the user to easily find the exact output directory corresponding to contigs 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.
        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)


#----------------------------------------------------
CONFIG FILE EDITING

Lines in the maker control files have the format key:value whith no spaces before or after the colon(:).  If the value is a file name, you can use relative paths and environmental variables, i.e. genome:$HOME/my_genome.fasta


MAKER has 3 control files for configuration options. A fourth file evaluator.ctl is used to supply a MAKER related program EVALUATOR with options specific to that program (only important if 'evaluate' is set to 1 in maker_opts.ctl).

Note that for all control files the comments written to help users begin with a pound sign(#).  In addition, options before the colon(:) can not be changed, nor should there be a space before or after the colon.

A. maker_exe.ctl - includes information about programs executed by MAKER.
Here an example of a section of the maker_exe.ctl file:
====================================
#-----Location of Executables Used by Maker/Evaluator
formatdb:/usr/local/bin/formatdb                              #location of NCBI formatdb executable
blastall:/usr/local/bin/blastall                              #location of NCBI blastall executable
xdformat:/usr/local/bin/xdformat                              #location of WUBLAST xdformat executable
blastn:/usr/local/bin/blastn                                  #location of WUBLAST blastn executable
blastx:/usr/local/bin/blastx                                  #location of WUBLAST blastx executable
tblastx:/usr/local/bin/tblastx                                #location of WUBLAST tblastx executable
RepeatMasker:/home/cholt/usr/local/RepeatMasker/RepeatMasker  #location of RepeatMasker executable
exonerate:/home/cholt/usr/local/exonerate/bin/exonerate       #location of exonerate executable

#-----Ab-initio Gene Prediction Algorithms
snap:/home/cholt/usr/local/snap/snap                  #location of snap executable
gmhmme3:/home/cholt/usr/local/gmes/gmhmme3            #location of eukaryotic genemark executable
augustus:/home/cholt/usr/local/augustus/bin/augustus  #location of augustus executable
fgenesh:/home/cholt/usr/local/fgenesh/fgenesh         #location of fgenesh executable

====================================


B. maker_bopts.ctl - contains statistics for fltering blast and exonerate data
Here an example of a section of the maker_bopts.ctl file:
====================================

#-----BLAST and Exonerate statistics thresholds
blast_type:wublast    #set to 'wublast' or 'ncbi'

pcov_blastn:0.8       #Blastn Percent Coverage Threhold EST-Genome Alignments
pid_blastn:0.85       #Blastn Percent Identity Threshold EST-Genome Aligments
eval_blastn:1e-10     #Blastn eval cutoff
bit_blastn:40         #Blastn bit cutoff

pcov_blastx:0.5       #Blastx Percent Coverage Threhold Protein-Genome Alignments
pid_blastx:0.4        #Blastx Percent Identity Threshold Protein-Genome Aligments
eval_blastx:1e-06     #Blastx eval cutoff
bit_blastx:30         #Blastx bit cutoff

pcov_rm_blastx:0.5    #Blastx Percent Coverage Threhold For Transposable Element Masking
pid_rm_blastx:0.4     #Blastx Percent Identity Threshold For Transposbale Element Masking
eval_rm_blastx:1e-06  #Blastx eval cutoff for transposable element masking
bit_rm_blastx:30      #Blastx bit cutoff for transposable element masking
====================================


C. maker_opts.ctl - contains options for maker and external programs used by maker
Here an example of a section of the maker_opts.ctl file:
====================================
#-----Genome (Required for De-Novo Annotations)
genome:input/genome.fasta  #genome sequence file in fasta format

#-----Re-annotation Options
genome_gff:     #re-annotate genome based on this gff3 file
est_pass:0      #use ests in genome_gff: 1 = yes, 0 = no
altest_pass:0   #use alternate organism ests in genome_gff: 1 = yes, 0 = no
protein_pass:0  #use proteins in genome_gff: 1 = yes, 0 = no
rm_pass:0       #use repeats in genome_gff: 1 = yes, 0 = no
model_pass:0    #use gene models in genome_gff: 1 = yes, 0 = no
pred_pass:0     #use ab-initio predictions in genome_gff: 1 = yes, 0 = no
other_pass:0    #passthrough everything else in genome_gff: 1 = yes, 0 = no

#-----EST Evidence (you must provide a value for at least one)
est:input/est.fasta        #non-redundant set of assembled ESTs in fasta format (classic EST analysis)
est_reads:                 #un-assembled EST reads in fasta format (for deep nextgen mRNASeq)
altest:input/altest.fasta  #EST/cDNA sequence file in fasta format from an alternate organism
est_gff:                   #EST evidence from a seperate gff3 file
altest_gff:                #Alternate organism EST evidence from a seperate gff3 file

#-----Protein Homology Evidence (you must provide a value for at least one)
protein:input/protein.fasta  #protein sequence file in fasta format
protein_gff:                 #protein homology evidence from a gff3 file
====================================

#----------------------------------------------------
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:input\est.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 genome_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.). 

#----------------------------------------------------
ADDING UTRs for GBROWSE

* When using APOLLO to visualize gene annotations, UTRs are inferred based on exon and CDS locations.  However GMOD and GBROWSE do not infer the UTR, so to visualize the UTR, you will have to run: add_utr_gff.pl with the following command:

maker2zff.pl <directory>
<directory> is the directory where all of your GFF files are located

each GFF file will have a sister file called sequence.wutr.gff3


#----------------------------------------------------
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/Apollo/ directory.  The file should be copied to the conf/ sub_directory which is located under the Apollo instalation directory.  Using the Mac version of Apollo the conf/ directory is located at /Applications/Apollo.app/Contents/Resources/app/conf/.


#----------------------------------------------------
HMM BUILDING (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> is 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.

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 documation (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