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SyMAP 3.4 System Guide SyMAP 3.4 System Guide System Guide This guide provides instructions for installing SyMAP, running the demo, and creating new projects. Please send bug reports and suggestions to symap@agcol.arizona.edu. Contents. Overview SyMAP (Synteny Mapping and Analysis Program) is a software package for detecting and displaying syntenic relationships between sequences.

SyMAP is very useful for comparative genomics as it can automatically generate and display comparisons between any number of organisms via an easy-to-use interface. The organisms can be represented either by sequenced chromosomes (pseudomolecules) or by FPC physical maps with sequenced markers and/or BESs. SyMAP is typically run as a standalone desktop application. It can also optionally generate a on systems with the required software packages. SyMAP generates several types of interactive displays, shown below.

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The top three are Java and the lower three are CGI/Perl: For details on the algorithm see: C. Soderlund, W. Shoemaker and A. Paterson (2006) SyMAP: A System for Discovering and Viewing Syntenic Regions of FPC maps Genome Research 16:1159-1168.

For information about the new additions in this version: C. Soderlund, M.

Bomhoff, and W. Nelson (2010) SyMAP: A turnkey synteny system with application to plant genomes. Manuscript submitted. The SyMAP User Agreement requires that one or both articles be cited if you use SyMAP in a paper, poster, or presentation. Before creating your own project, we strongly recommend you run the demo.

The demo requires NO installations except downloading SyMAP. BEWARE: If you will be executing SyMAP on the recent MAC, there may be are problems with the 3D Java library. That is, everything should run fine until you try opening the 3D display. Installing SyMAP SyMAP can be run on Intel systems with Linux or Mac-OSX, and Windows is supported for the viewing functions only. Version 5 or later is required both for the viewing and for the alignment functions. To run the demo, and perform alignments in general, we recommend a system with a minimum of two processors and 4GB RAM.

SyMAP uses one processor by default, but the demo will run fastest using two processors; the number of processors used is set by the '-p' command line parameter. A minimum of 2GB RAM per processor is recommended, although small genomes (e.g., bacterial) need less.

For large chromosomes, e.g. Mammalian, MUMer can use up to 10GB per processor, and you will also need to run on a 64-bit computer.

Note that SyMAP uses 2GB by default, so if your computer has less than this amount, you will need to reduce SyMAP's memory setting (see the guide). Extract the package into a convenient location, using the following command (on a Mac, you will need to work in a Unix terminal window.) tar -xzvf symap33.tar.gz A symap33 directory is created containing all of the SyMAP program and data files. You are now ready to run the demo. Running the Demo SyMAP comes with a demo dataset (derived from the rice and sorghum genomes, and the maize FPC map).

It is highly recommended to run through the demo to make sure everything is working, before beginning on your real dataset. As mentioned, computing synteny only runs on Mac or Linux. If you have MySQL installed, then edit the file symap.config to enter the database name, host, user and password (see 'Creating a New Project' for more information). If you do not have MySQL installed, do not edit the file symap.config; SyMAP will create the database for you with the MySQL program supplied with the package. Using the SyMAP supplied MySQL is suitable for small projects, but for large projects or to show results on the web, you should install MySQL.

Note: do not run SyMAP as the Linux root user (MySQL will not start). Use a regular user account.

Go to the symap33 directory and run SyMAP: cd symap33./symap The alignment for moderate-sized chromosomes, such as those used in this demo, requires a minimum of 2 GB RAM per processor. For large chromosomes, e.g. Maize or human, up to 10 GB per processor may be needed, in order for MUMmer to build the suffix tree for the target pseudomolecule. Performance is severely degraded if your system does not have adequate memory. If you wish to use more than one processor (recommended to speed the alignment process), use the '-p' option, e.g./symap -p 2 For the demo there is no benefit to using more than two (but for an alignment with more than two chromosomes on the target side, there would be a benefit as long as memory meets the above guidelines). If you did not specify a database for SyMAP, you will see output to the terminal as the database starts up, such as: MysqldResource launching mysqld (driverlaunchedmysqld1) InnoDB: The first specified data file./ibdata1 did not exist: InnoDB: a new database to be created! 090624 16:35:19 InnoDB: Setting file./ibdata1 size to 10 MB.

This output can be ignored. The Project Manager window will then appear:. Select the demoseq and demoseq2 projects listed on the left. The projects will appear listed on the right with additional summary information and options.

Load the two selected projects by clicking the links that say Project not loaded, click to load to load it into the SyMAP database. A popup window appears showing the progress; click 'Done' when it finishes. The projects that are loaded now appear in the 'Available Alignments' table. Select the square for demoseq to demoseq2 in the table, and click the 'Align' button below to align the projects. The alignment will take about 30 minutes to complete (if two processors are used). A popup window shows progress; click Done when it is finished. The progress printouts are also saved to a log file symap.log, which is located under the project pair directory, data/pseudopseudo/demoseqtodemoseq2.

In addition, the blocks and the synteny anchors are listed in tabular ('csv') format in the /results subdirectory of the project pair directory. Now that the projects are aligned, an 'x' appears in the (Chr1, Chr3) square of the table. The displays are now available; for example, click the 'Show Dot Plot' button to see that display: Try the following:. Click the cell in the upper left (chromosome 3 to chromosome 1) to zoom in.

Click one of the synteny blocks (blue rectangles) to highlight it, then click again to show that synteny block region in the 2D Alignment Display. See the for more information on these displays. Click the 'Show 3D' button to view the 3D Alignment Display.

Look at the upper left side, under 'Demo-Seq'. You will see that chromosome 3 has a red box around its number. This means that it is the 'reference sequence' which others will align to.

You can change reference by clicking the numbers. To see the synteny of other chromosomes to the reference, click on their bar ( not their number). Clicking on chromosome 1 and 3 under 'Demo-Seq2' gives the picture shown below: Clicking the '2D' button at the bottom left brings up a 2D version of this same set of alignments. For more on this display, see the. Note that when you go from the 3D to the 2D view, only the 'synteny hits' will be shown (these are the anchors which are part of a synteny block). You can change this using the 'Hit Filter' menu in the 2D view.

FPC to sequence synteny: Repeat steps 2-6 using the demofpc and demoseq1 projects. Everything is the same, except for the appearance of the 2D display, which shows an FPC contig. Creating a New Project To create your own project, you first have to choose which database to use:. Use the built-in MySQL: The simplest, as it is already set up. However, this database cannot be accessed from the web, and it is not configured for very large projects. Create your own MySQL database: In this case, (1) MySQL must be installed, and (2) you must provide the server, user and password for the MySQL in the main symap.config file. The file is in the symap33 directory, and uses the format dbname = dbserver = dbadminuser = dbadminpasswd = The program will create a MySQL database named dbname on the specified host dbserver.

If you get database errors, see. Use the following instructions to load your own data into SyMAP.

Create the project directories. The projectname is a short but descriptive name that you choose for the project, such as the species name 'rice' or 'maize'.

It can contain letters, numbers, and underscores. For a pseudomolecule (sequence) project, create the following directory structure: symap33/data/pseudo/ projectname/ annotation/ (optional) sequence/ pseudo/ For an FPC project, create the following directory structure: symap33/data/fpc projectname/ annotation/ (optional) sequence/ bes/ mrk/.

Import your data. For a pseudomolecule project:. Copy the FASTA sequence files into the sequence/pseudo/ directory. Note: there must only be one section per FASTA file, with the section header in the format ChrX where X is a number or character. Do not use Chr0 as a name. See Step 3 for more naming options.

(Optional) Copy your GFF3 annotation files into the annotation/ directory. SyMAP recognizes annotations of type gene, exon, CDS, centromere, and gap ('type' is column 3 in the gff file; type CDS is converted to exon).

Also, SyMAP reads 'keyword=value' pairs from the attribute field (last column), which will be displayed as the 'Gene description'. To include one or more links to another site, use 'name=URL', where the gene description will then include the 'name' linked to the site. For an FPC project:. Make sure the BES are named as + (e.g., if clone is a0003B01, BES can be a0003B01f or a0003B01r). Otherwise, SyMAP cannot connect the BES to its clone. Copy the BES FASTA sequence files into the sequence/bes/ directory.

Copy the marker FASTA sequence files into the sequence/mrk/ directory. Copy the FPC file into the projectname/ directory. Copy your GFF3 annotation files (if any) into the annotation/ directory.

See the description of the GFF3 annotation file above. Set the project parameters in the projectname/params file.

Use the demo params files shown below as templates. For pseudomolecule project: symap33/data/pseudo/demoseq1/params: displayname = Demo-Seq1 # name for display grpprefix = chr grporder = 1,2,3,4,5,6,7,8,9,10 # these can include letters or letter/number mixtures If the sequences contain characters other than agctnAGCTN, MUMmer will not accept them. You can either edit them yourself, or use the provided script fixSeq.pl: perl fixSeq.pl file1 file2. The script creates fixed sequence files with suffix '.fix' appended. If the sequences are unordered, leave out grporder, and use: grpsort = unordered Unordered sequences can be all in one fasta file (but they don't have to be).

For unordered sequences, there are also two more optional parameters: orderagainst = minsize = The first allows you to specify a reference project which will be used to order the scaffolds. They will be ordered according to their largest synteny blocks, when the alignment to the reference project is run. This ordering will then be used in the dot plot displays against other projects.

Note that the reference project name should be the directory name under which the project is is located, i.e. The minsize parameter controls the size of scaffolds which are loaded. The default is 100kb, i.e., scaffolds at least 100kb will be loaded from the fasta file.

If you wish to use the gene-masking feature to mask out all but genes, add the parameter usegenemask=1. For FPC project: symap33/data/fpc/demofpc/params: cbsize = 4096 # average band size in base pairs displayname = Demo-FPC # name for display grpprefix = chr grporder = 1,2,3,4,5,6,7,8,9,10 # these can include letters or letter/number mixtures. Load the new project into SyMAP.

First, run the SyMAP program from the symap33 directory:./symap The Project Manager window will appear. SyMAP reads the project directories and lists them on the left-side of the window, so your project will automatically be listed. When selected, the project summary will be shown on the right side.

Click the 'load project' link on the right. A progress window will appear while the project data is loaded into the database. Align and display the new project against any other existing project by following the example in the demo. Most alignments require 2GB RAM per processor, but the largest chromosomes may require more, and a 64-bit machine as well. The table below shows examples of time and memory needs for various alignments: Alignments Max. RAM Used Total Time.

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Maize Sequence (2Gb; repeat-masked) to Rice (372 Mbp) 400 MB 10 hrs Maize FPC (614 Mbp) to Rice (372 Mbp) 400 MB 5 hrs Rice (372 Mbp) to Sorghum (660 Mbp) 8 GB 11 hrs. On a system with 4 dual-core 3.66 Ghz processors. Parameters SyMAP typically provides good results without adjusting parameters, but there are several which can be adjusted to fine-tune the synteny analysis. The easiest way to set parameters is to add then to the main parameter file symap.config, in which case they apply to all subsequent processing. You can also add them to the params file for just one species pair.

To do this, it is easiest to first run the alignment with default parameters, then do the following:. Locate the pair directory a data/pseudopseudo/ project1to project2 (or data/fpcpseudo, for an FPC alignment). This was created when the alignment was started.

Create a params file within the directory and add the desired parameters. Then, re-run the alignment, choosing to use the MUMmer or BLAT files which were already created. Following are the most relevant parameters:.

mindots allowed values = 3, default 7 The number of anchors required to form a synteny block. Raise/lower this number to decrease/increase the number of blocks found. Note, a number of quality criteria are also applied during the block finding process (see Reference 5). topn allowed values = 0, default 2 Anchors are filtered before loading to the database. By default, the top 2 hits for each query are retained (see Reference 5).

Raise/lower this value to increase/decrease the number of hits retained. Set to 0 to disable filtering altogether. mergeblocks allowed values 0,1, default 0 Merging blocks across large gaps can improve the clarity of block-based displays such as the CGI web views supplied with SyMAP (see Reference 5).

In previous version of SyMAP, blocks were merged by default but this has been changed since the newer chromosome-based displays are clearer without it. dosynteny allowed values 0,1, default 1 For some highly repetitive alignments, particularly self alignments, the synteny algorithm may take time to run and it may be desirable to see the dotplot without computing blocks first. Set dosynteny=0 in this case. doclustering allowed values 0,1, default 1 Anchors are clustered prior to loading, as described in Reference 6. To disable this (and see the individual MUMmer alignments), set doclustering=0. This can increase processing time and memory usage greatly for large sequence projects!. usegenemask allowed values 0,1, default 0 If this option is set, the software will generate a version of the pseudomolecules having all but genes masked out with N's (assuming that gene annotation is present).

This reduces the computation time for large genomes (using a repeat-masked genome is another option). It is easiest to set this parameter in the main parameter file symap.config, before loading the pseudomolecules and doing alignments. However, it will then be used for all alignments, so be sure to comment the parameter out when doing alignments where you don't want masking. (You can also set it in the params file for the genome you want to mask, and in the separate params files for the alignment pairs you want to use the masking in.) If you have already loaded the sequences, use 'reload annotation' to cause the gene-masked files to be written. nooverlappingblocks allowed values 0,1, default 1 SyMAP is designed to find all blocks, including those from duplication events. If you only want to see the best block covering each sequence region, set this parameter to 1.

The best block is defined as the one having the most anchors. For example, for the alignment of rice to soy, one could create the following file named params with the following values: mindots = 10 topn = 4 and place it in the directory data/pseudopseudo/ricetosoy. Increasing the memory limit For large projects, especially self-alignments, with a million or more raw hits, SyMAP may need more than the default 2G RAM to process the data (although we have not found this necessary, even for projects having over 20M anchors). See for instructions on how to increase the amount of memory available to SyMAP. Using NUCmer rather than PROmer By default SyMAP uses the PROmer mode of MUMmer (aligning by translated reading frames) except for self-alignments which default to NUCmer (nucleotide alignment) to reduce the very large output. To specify whether PROmer or NUCmer is used, edit the first line in the alignparams file in the project pair directory (same location as the params file above) accordingly: program:nucmer type1:pseudo type2:pseudo args:' Additional arguments may also be specified for PROmer, NUCmer, or BLAT using the 'args' field.

Web Applet The optional web interface requires these external packages:. Web server software (e.g. version 5 or later.

Perl with the following CPAN modules: DBI Cwd Data::Page Storable File::Temp CGI CGI::Carp GD Imager Math::Bezier. Make sure that you have MySQL running on the server. MySQL must be configured with accessible admin and user accounts (the admin account is used to load data, the user account is used to display data to the web). Test your account settings by trying to connect from a client system: mysql -h -u -p. Use the Project Manager to create, load and align the genomes, as described in 'Creating a New Project'.

In the symap/ directory edit the symap.config file. The symap.config file specifies the database location and login information. It also specifies the locations for web files in the local file system and the corresponding URLs for those files on the internet (your web server must already by configured accordingly). Edit the required parameters listed below in the symap.config file for your system configuration. SyMAP will automatically create the database.

Parameter Description dbname (optional) The name of the database to create, or blank to use 'symap'. Dbserver (required) The name of the MySQL database server. Dbadminuser (required) The name of the 'admin' database user with table create privileges.

Dbadminpasswd (required) The password for the 'admin' user. Dbclientuser (required) The name of the 'client' database user (only requires read access). Dbclientpasswd (required) The password for the 'client' user. Htmlpath (required) The file system path to install the HTML files.

Cgipath (required) The file system path to install the CGI files. Htmlurl (required) The URL path to the htmlpath directory. Cgiurl (required) The URL path to the cgiurl directory. Logfile (optional) The name to use for the log file. Sitelogo (optional) The path and file name for the site logo image.

Example: dbname = mysymapdatabase dbserver = myserver.mydomain.edu dbadminuser = admin dbadminpasswd = dbclientuser = client dbclientpasswd = htmlpath = /web/htdocs/symap cgipath = /web/cgi-bin/symap htmlurl = cgiurl =. Install web files. perl scripts/install.pl This command copies the SyMAP program files into the 'htmlpath' and 'cgipath' directories specified in the symap.config file. The output should look like this: executing: rm -rf /web/htdocs/symap executing: rm -rf /web/cgi-bin/symap executing: cp -fr html /web/htdocs/symap executing: cp -fr java/jar/. /web/htdocs/symap executing: cp -fr java/src/properties /web/htdocs/symap executing: chmod 777 /web/htdocs/symap/png executing: chmod 777 /web/htdocs/symap/jpeg executing: cp -fr cgi /web/cgi-bin/symap executing: cp -f symap33/symap.pm /web/cgi-bin/symap/symap.pm SyMAP web installation complete. To view, go to Note: if an error occurs at any point in this process, remove the symap installation from the disk (and remote database if applicable) and re-install the SyMAP package. References 1 Kent, J.

(2002) BLAT-the BLAST-like alignment tool, Genome Research 12:656-64. 2 Kurtz, S., Phillippy, A., Delcher, A.L., Smoot, M., Shumway, M., Antonescu, C., Salzberg, S.L. (2004) Versatile and open software for comparing large genomes, Genome Biology, 5:R12 3 Holland, R., T. Schreiber (2008) BioJava: an Open-Source Framework for Bioinformatics Bioinformatics 2008; doi: 10.1093/bioinformatics/btn397 4 Krzywinski, M., J. Marra (2009) Circos: An information aesthetic for comparative genomics. Genome Research doi:10.1101/gr.092759.109. 5 Soderlund, C., Nelson, W., Shoemaker, A., and Paterson, A.(2006) SyMAP: A system for discovering and viewing syntenic regions of FPC maps.

6 Soderlund, C., Bomhoff, M., and Nelson, W. SyMAP: A turnkey synteny system with application to multiple large duplicated plant sequenced genomes. Manuscript submitted. Email Comments To.