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.  MAKER works on both eukaryotic and prokaryotic genomes.


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-ES (exon.biology.gatech.edu)
    *FGENESH 2.6 or higher (www.softberry.com) - requires licence
    *GeneMarkS for prokaryotic genomes (exon.biology.gatech.edu)

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

Also see the mpi_maker installation instructions found further bellow.


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 -zxvf 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.
    perl   - contains perl modules that need to be compiled

Finally change to the maker/perl directory and type: 'perl Install.PL'
to compile required perl modules.

Now you can run MAKER!!

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

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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 for your OS (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 4 nodes or processors)

mpiexec -n 4 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.

#---------------------------------------------------------------------

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 seperate
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 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.


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 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

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 is an example of sections of 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.

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 THE 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.


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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).


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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.).

You should use the online GFF3 validator to see if your GFF3 files
comply with all GFF3 specifications before running MAKER:

     http://dev.wormbase.org/db/validate_gff3/validate_gff3_online

#---------------------------------------------------------------------

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:

     add_utr_gff.pl <directory>

     <directory> is the directory containing all of your GFF3 files.

Each GFF3 file will have a sister file called sequence.wutr.gff3.

There is also another script called add_utr_start_stop that adds start
and stop codon entries as well as UTR entries.

     add_utr_start_stop <GFF3 file>

#---------------------------------------------------------------------

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> 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.

   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