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LAVA release 0.1.3

This update allows compatibility with the latest primer3 version, in which the thermodynamic parameters have been embedded as part of the code instead of as accessory files. As a result, the thermodynamic path command line argument is no longer needed.

LAVA release 0.1.2 (by Michael Bekaert [email protected])

Lava has been updated to be compatible with any Linux distribution. Part of the source code was updated to allow more parameters to be accesible outer_pair_target_length, middle_pair_target_length and inner_pair_target_length.

LAVA release 0.1.1 (by Michael Bekaert [email protected])

Lava has been updated to be compatible with the lasted update of Primer3 and BioPerl. Part of the source code was updated to fix syntax errors and deprecated code. A new mandatory parameter as been added PRIMER_THERMODYNAMIC_PARAMETERS_PATH, which should provide the path to the primer3_config directory. Usage --thermodynamic_path=path or <thermodynamic_path>path</thermodynamic_path>

SYSTEM REQUIREMENTS

Primer3 2+ (available at http://primer3.sourceforge.net/) Primer-selection code

BioPerl 1.6+ (available at http://www.bioperl.org/) BioPerl's fundamental utilities and file IO are used heavily

PRIMER3 REQUIRMENTS

PRIMER_THERMODYNAMIC_PARAMETERS_PATH must point to the right location. This tag specifies the path to the directory that contains all the parameter files used by the thermodynamic approach. In Linux, there are two default locations that are tested if this tag is not defined: ./primer3_config/ and /usr/local/share/primer3/primer3_config/. For Windows, there is only one default location: .\primer3_config. If the the parameter files are not in one these locations, be sure to set PRIMER_THERMODYNAMIC_PARAMETERS_PATH.

So if you download and compile primer3 form source using the Make command, to get primer3 to run globally you need to copy the executueable, primer3_core, to your path and place the configuration directory, primer3_config in that same directory or at /usr/local/share/primer3/primer3_config/

cd src sudo cp primer3_core /usr/local/bin sudo mkdir -p /usr/local/share/primer3/ sudo cp -r primer3_config /usr/local/share/primer3/primer3_config/

LAVA release 0.1 (by Clinton Torres [email protected])

Original distribution and algorithm

INTRODUCTION

LAVA (LAMP Assay Versatile Analysis) is an extendable open-source Loop-mediated isothermal AMPlification (LAMP). LAVA identifies combinations of six primer regions for basic LAMP signatures, or combinations of eight primer regions for LAMP signatures with loop primers, which can be used as LAMP signatures. The identified primers are conserved among target organism sequences. Primer combinations are optimized based on lengths, melting temperatures, and spacing among primer sites.

This is a Multiple-Sequence Alignment (MSA) approach to LAMP signature design.
Either a single sequence or a pre-computed MSA can be used as input. The individual sequences from the MSA are used for primer enumeration, with the goal of identifying as many locations for primers as possible. Design parameters can be adjusted for each primer role. Once primers have been identified that are shared among all the individual sequences, they are combined into potential inner primer pairs. Finally, combinations of primers for the remaining roles of loop, middle, and outer, are analyzed to select LAMP signature candidates.

The script slava.pl is also included as a demonstration of scaling to larger genome sizes, but with this kind of implementation, the processing requirements scale with the sequence size. It's realistic to expect that a bacteria-sized genome will take hours to days to execute, depending on the computing resources available.

SYSTEM REQUIREMENTS

This is a Unix/Linux-only source code release. It is not unreasonable to assume that the source code can be installed and run on other system types, with a minimal amount of effort spent porting, especially since it is primarily implemented in Perl.

Perl library XML::LibXML (available at http://www.cpan.org/) XML::LibXML is used for some of Options.pm's persistance capabilities

BioPerl 1.5.2 (available at http://www.bioperl.org/) BioPerl's fundamental utilities and file IO are used heavily

Primer3 1.0 (available at http://primer3.sourceforge.net/) Primer-selection code

Bio::Perl::Run (also available at http://www.bioperl.org/ as "bioperl-run") Bio::Perl::Run::Primer3 is used to execute system-level calls of Primer3

INSTALLATION INSTRUCTIONS

There are two components to this program, the script lava.pl, and the Perl modules that the script relies on. Installation of the modules can be performed by entering the top-level directory of the distribution and running:

$ perl Makefile.PL $ make $ make test $ make install

After the Perl modules are installed, the script lava.pl can be run from anywhere that the installed Perl modules are in the @INC path.

ACKNOWLEDGEMENTS

This work was supported by a NIBIB Point-of-Care Technologies Research Network Center grant (Gerald J. Kost, PI of the UC Davis / LLNL POCT Center, NIH U54 EB007959). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Biomedical Imaging and Bioengineering or the National Institutes of Health.

This work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.