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gmap-gsnap
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0.  Availability
============

The source code for this package is available from
http://research-pub.gene.com/gmap.  License terms are provided in the
COPYING file.


1.  Building and installing GMAP and GSNAP
==========================================

Prerequisites: a Unix system (including Cygwin on Windows), a C
compiler, and Perl

Step 1: Set your site-specific variables by editing the file
config.site.  In particular, you should set appropriate values for
"prefix" and probably for "with_gmapdb", as explained in that file.
If you are compiling this package on a Macintosh, you may need to edit
CFLAGS to be

CFLAGS = '-O3 -m64'

since Macintosh machines will make only 32-bit executables by default.


Step 2: Build, test, and install the programs, by running the
following GNU commands

    ./configure
    make
    make check   (optional)
    make install

Note 1: Instead of editing the config.site file in step 1, you may type
everything on the command line for the ./configure script in step 2,
like this

    ./configure --prefix=/your/usr/local/path --with-gmapdb=/path/to/gmapdb

If you omit --with-gmapdb, it defaults to ${prefix}/share.  If you
omit --prefix, it defaults to /usr/local.  Note that on the command
line, it is "with-gmapdb" with a hyphen, but in a config.site file,
it is "with_gmapdb" with an underscore.


Note 2: If you want to keep your version of config.site or have
multiple versions, you can save the file to a different filename, and
then refer to it like this

    ./configure CONFIG_SITE=


Note 3: GSNAP is designed for short reads of a limited length, and
relies upon a maximum read length variable MAX_READLENGTH defined at
compile time (default 250).  You may set this variable by providing it
to configure like this

    ./configure MAX_READLENGTH=

or by defining it in your config.site file (or in the file provided to
configure as the value of CONFIG_SITE).  Or you may set the value of
MAX_READLENGTH as an environment variable before calling ./configure.
If you do not set MAX_READLENGTH, it will have the default value shown
when you run "./configure --help".

Note that MAX_READLENGTH applies only to GSNAP.  GMAP, on the other
hand, can process queries up to 1 million bp.


Note 4: GSNAP can read from gzip-compressed FASTA or FASTQ input
files.  This feature requires the zlib library to be present
(available from http://www.zlib.net).  The configure program will
detect the availability of zlib automatically.  However, to disable
this feature, you can add "--disable-zlib" to the ./configure command
or edit your config.site file to have the command "disable_zlib".


Note 5: GSNAP optionally supports the Goby input and output file
formats.  To implement this functionality, you need to obtain and
compile the libraries from http://campagnelab.org/software/goby.  If
the resulting header files are located in /path/to/goby/include and
the library files are in /path/to/goby/lib, you can then add the flag
"--with-goby=/path/to/goby" to your ./configure command or edit your
config.site file to have this directory as the value for "with_goby".



2.  Downloading a pre-built GMAP/GSNAP database
===============================================

A GMAP/GSNAP "database" is a set of genomic index files, representing
the genome in a hash table format.  You can use the programs
gmap_build or gmap_setup to build your own database (as described
below), but you can started quickly by downloading a pre-built
GMAP/GSNAP database from the same place you obtained the GMAP program
(see above for URL).

Place the database in the GMAPDB directory you specified in the
config.site file when you built the gmap program.  You should include
a subdirectory for each GMAP database; for example, if you downloaded
a database called , your directory structure should look like
this

    /path/to/gmapdb//
    /path/to/gmapdb//.chromosome
    /path/to/gmapdb//.chromosome.iit
    ...
    /path/to/gmapdb//.version

Note that the GMAP database format has changed with the 2011-08-15
release.  Older versions of GMAP and GSNAP will not work with the
newer databases, but the current version of the programs is backward
compatible with the older databases.  Also, versions of GMAP and GSNAP
before 2008 may require symbolic links to work even with the older
databases.

The old databases have the index files .ref3offsets and
.ref3positions.  The new databases have the index files
.ref12153gammaptrs, .ref12153offsetscomp, and
.ref153positions, if built using a base size of 12, a k-mer
size of 15, and skipping every 3 bp in the genome.  If the k-mer size
is equal to the base size, then the gammaptrs file will be absent.

Also, the name of the positions file has changed starting with
2012-02-14 version.  Previously, the file was named
.ref12153positions, but it is now named
.ref153positions, since the contents are independent of the
base size.  If you create a database with a newer version of the
package, and want older versions of the GMAP or GSNAP to work with
these newer versions, you will need to make a symbolic link like this:

    ln -s .ref153positions .ref12153.positions



3.  Setting up to build a GMAP/GSNAP database (one chromosome per FASTA entry)
==============================================================================

You can also build your own genomic database, using one of two utility
programs provided with this package: gmap_build (the newer, one-step
method) or gmap_setup (the older way that uses Makefile and requires
multiple steps).  Note that the total sequence length in your database
cannot exceed 2^32 = 4,294,967,296 (about 4 billion) bp.  This is
because the format uses 32-bit pointers.  If your total sequence
provided to the utility programs exceeds 4 billion bp, the programs
will abort.  Below I use the "genome" and "chromosome", but the input
sequences can be anything you wish to align to, including transcripts
or small fragments.

You will need to start with a set of FASTA files containing either
entire chromosomes or contigs that represent pieces of chromosomes.
If your FASTA entries each contain a single chromosome, and the
accession for each chromosome is the chromosome number/letter, you can
simply run this command

    gmap_build -d  [-k ] 

which will build and install the GMAP index files in your default
GMAPDB location.

You can see the full usage of gmap_build by doing "gmap_build --help",
but here are some useful flags.  If your FASTA files are gzipped, you
can add the flag "-g" to gmap_build.  You can control the k-mer size
for the genomic index with the -k flag, which can range from 12 to 15.
The default value for -k is 15, but this requires your machine to have
4 GB of RAM to build the indices.  If you do not have 4 GB of RAM,
then you will need to reduce the value of -k or find another machine.
Here are the RAM requirements for building various indices:

    k-mer of 12: 64 MB
    k-mer of 13: 256 MB
    k-mer of 14: 1 GB
    k-mer of 15: 4 GB

These are the RAM requirements for building indices, but not to run
the GMAP/GSNAP programs once the indices are built, because the
genomic indices are compressed.  For example, the genomic index for a
k-mer of 15 gives a gammaptrs file of 64 MB and an offsetscomp file of
about 350 MB, much smaller than the 4 GB that would otherwise be
required.  Therefore, you may want to build your genomic index on a
computer with sufficient RAM, and distribute that index to be used by
computers with less RAM.

If you want to build your genomic databases with more than one k-mer
size, you can re-run gmap_build with different values of -k.  This
will overwrite only the identical files from the previous runs.  You
can then choose the k-mer size at run-time by using the -k flag for
either GMAP or GSNAP.



4.  Setting up to build a GMAP/GSNAP database (more complex cases)
==================================================================

If gmap_build works for you, you can skip to section 5.  Otherwise, if
you have more complicated needs than gmap_build can handle, there is a
more general build tool called gmap_setup, which creates a Makefile
with this command

    gmap_setup -d  [-k ] ...

and then has you run a few make commands, based on the directions it
provides.  Again, you can type "gmap_setup --help" to see the full set
of options.

Note that the term

    ... 

above indicates that multiple files can be listed.  The files can be
in any order, and the contigs can be in any order within these files.
By default, the GMAP setup process will sort the contigs and
chromosomes into their appropriate "chrom" order.  For the human
genome, this order is 1, 2, ..., 10, 11, ..., 22, X, Y, M, followed by
all other chromosomes in numeric/alphabetical order.  If you don't
want this sort, provide the "-s none" flag to gmap_setup or
gmap_build.  Other sort options besides "none" and "chrom" are "alpha"
and "numeric-alpha".

We show the full set of gmap_setup options under item 4d below, but
we first discuss some specific situations for using the program.


4a.  Chromosomes represented as contig pieces
=============================================

If your FASTA entries consist of contigs, each of which has a mapping
to a chromosomal region in the header, you may need to add the -C flag
to gmap_setup, like this

    gmap_setup -d  -C ...

Then gmap_setup will try to parse a chromosomal region from each
header.  The program knows how to parse the following patterns:

    chr=1:217281..257582  [may insert spaces around '=', or omit '=' character]
    chr=1                 [may insert spaces around '=', or omit '=' character]
    chromosome 1                                                         [NCBI .mfa format]
    chromosome:NCBI35:22:1:49554710:1                                    [Ensembl format]
    /chromosome=2                                                        [Celera format]
    /chromosome=2 /alignment=(88840247-88864134) /orientation=rev        [Celera format]
    chr1:217281..257582
    chr1                  [may insert spaces after 'chr']

If only the chromosome is specified, without coordinates, the program
will assign its own chromosomal coordinates by concatenating the
contigs within each chromosome.  If gmap_setup cannot figure out the
chromosome, it will assign it to chromosome "NA".


4b.  Using an MD file
=====================

Another possibility is that your FASTA entries consist of contigs,
each of which has mapping information in an external file.  Genomes
from NCBI typically include an ".md" file (like seq_contig.md) that
specifies the chromosomal coordinates for each contig.  To use this
information, provide the -M flag to gmap_setup, like this

    gmap_setup -d  -M  ...

The program will then try to parse the mdfile (which often changes
formats) and verify with you which columns contain the contig names
and chromosomal coordinates.


4c.  Compressed FASTA files or files requiring processing
=========================================================

If your genome files don't satisfy any of the cases above, you may
need to write a small script that pipes the sequences in FASTA format
to gmap_setup.  You can tell gmap_setup about your script with the -E
flag, like this

    gmap_setup -d  -E 'gunzip -c chr*.gz'
    gmap_setup -d  -E 'cat *.fa | ./add-chromosomal-info.pl'

You can think of the command as a Unix pipe for processing each FASTA
file before it is read by gmap_setup.


4d.  General use of gmap_setup program
======================================

Any of the steps above (4a, 4b, or 4c) will create a Makefile, called
Makefile..  You will then use this Makefile to build a
GMAP/GSNAP database.  You will be prompted to use this Makefile
through the following commands:

    make -f Makefile. coords
    make -f Makefile. gmapdb
    make -f Makefile. install

Note that older versions of GMAP allowed the building of genomic
databases containing lower-case characters by doing "make -f
Makefile. gmapdb_lc" or "make -f Makefile.
gmapdb_lc_masked", but these will not work with GSNAP, and I am not
certain if these still work with the most recent GMAP either, so they
are not currently supported.


The first step in using this Makefile is to create a file called
coords..  You may manually edit this file, if you wish, before
proceeding with the rest of the Makefile steps.  The coords file
contains one contig per line, in the following format:

		

where the chromosomal_mapping is in the form
:...  Here are some examples:

NT_077911.1	1:217281..257582
NT_091704.1	22U:1..166566

If you want the contig to be inserted as its reverse complement, then
list the coordinates in the reverse direction (starting with the
higher number), like this:

NT_039199.1	1:61563373..61273712

You may delete lines or comment them out with a '#' character, which
will effectively omit those contigs from your genome build.  You may
also change chromosomal assignments (in column 2) at this stage.

Note: Previous versions of GMAP allowed you to specify alternate
strains in column 3, but this feature added too much complexity and is
no longer supported.


You then will run "make -f Makefile. gmapdb".  This creates a
compressed version of the genome, in the file .genomecomp,
which can hold only the standard, upper-case A, C, G, T, N, and X
characters.  It converts all lower-case characters to upper-case, and
all non-ACGTNX characters to 'N'.  This command also creates a hash
table of the genome, with files that end with "gammaptrs",
"offsetscomp", and "positions".

Finally, running "make -f Makefile. install" will place all
database files in a subdirectory specified by your "-d" flag under the
directory specified either by the "-D" flag or, if not specified, the
value of --with-gmapdb you provided at configure time.



Running GMAP
============

To see the full set of options, type "gmap --help".  The following are
some common examples of usage.  For more examples, see the document
available at http://www.gene.com/share/gmap/paper/demo-slides.pdf

For each of the examples below, we assume that you have installed a
genome database called  in your GMAPDB directory.  (If your
database is located elsewhere, you can specify the -D flag to gmap or
set the environment variable GMAPDB to point to that directory.)

* Mapping only: To map one or more cDNAs in a FASTA file onto a
  genome, run GMAP as follows:

    gmap -d  

* Mapping and alignment: If you want to map the cDNAs to a genome,
  and show the full alignment, provide the -A flag:

    gmap -d  -A 


* Alignment only: To align one or more cDNAs in a FASTA file onto a
  given genomic segment (also in a FASTA file), use the -g flag
  instead of the -d flag:

    gmap -g  -A 


* Batch mode: If you have a large number of cDNAs to run, and you have
  sufficient RAM to run in batch mode, add the "-B 3", "-B 4", or "-B
  5" option.  Details for these options are provided by running "gmap
  --help".

    gmap -d  -B 5 -A 


* Multithreaded mode: If your machine has several processors, you can
  make batch mode run even faster by specifying multiple threads with
  the -t flag:

    gmap -d  -B 5 -A -t  

  Note that with multiple threads, the output results will appear in
  random order, depending on which thread finishes its computation
  first.  If you wish your output to be in the same order as the 
  input cDNA file, add the '-O' (letter O, not the number 0) flag
  to get ordered output.

  Guidelines: The -t flag specifies the number of computational
  threads.  In addition, if your machine supports threads, GMAP also
  uses one thread for reading the input query sequences, and one
  thread for printing the output results.  Therefore, the total number
  of threads will be 2 plus the number you specify.  The program will
  work optimally if it uses one thread per available processor.  If
  you specify too many threads, you can cause your computer to thrash
  and slow down.  Note that other programs running on your computer
  also need processors.


* Compressed output: If you want to store the alignment results in a
  compressed format, use the -Z flag.  You can uncompress the results
  by using the gmap_uncompress.pl program:

    gmap -d  -Z  > x
    cat x | gmap_uncompress


Building map files
==================

This package includes an implementation of interval index trees
(IITs), which permits efficient lookup of interval information.  The
gmap program also allows you (with its -m flag) to look up pre-mapped
annotation information that overlaps your query cDNA sequence.  These
interval index trees (or map files) are built using the iit_store
program included in this package.  To build a map file, do the
following:

Step 1: Put your map information for a given genome into a map file
with the following FASTA-like format:
   
    >label coords optional_tag
    optional_annotation (which may be zero, one, or multiple lines)

For example, the label may be an EST accession, with the coords
representing its genomic position.  Labels may be duplicated if
necessary.

The coords should be of the form

    chr:position
    chr:startposition..endposition

The term "chr:position" is equivalent to "chr:position..position".  If
you want to indicate that the interval is on the minus strand or
reverse direction, then  may be less than
.

Tags are very general and can be used for a variety of purposes.  For
example, you could 


Step 2: Run iit_store on this map file as follows

    cat  | iit_store -o 

The program will create a file called .iit.

Now you can retrieve this information with iit_get

    iit_get .iit 

where  has the format "chr:position" or
"chr:startposition..endposition".  The iit_get program has other
capabilities, including the ability to retrieve information by label,
like this:

    iit_get .iit

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