P-SAMS

Plant Small RNA Maker Suite

Installing P-SAMS

Minimum Software Requirements

• Tested on Perl v5.10.1 built for x86_64-linux-thread-multi
• Required Perl modules
  • Config::Tiny
  • DBI
  • HTML::Entities
  • Data::Dumper
• TargetFinder v1.7 (https://github.com/carringtonlab/TargetFinder)
• MySQL (http://www.mysql.com/)
• Samtools v0.1.19+ (http://www.htslib.org/)

Optional Software

P-SAMS has the option to execute TargetFinder jobs on a batch system using the Terascale Open-source Resource and QUEue Manager (TORQUE; http://www.adaptivecomputing.com/products/open-source/torque/).

Installation

Clone the P-SAMS repository with the TargetFinder submodule.

git clone --recursive https://github.com/carringtonlab/p-sams.git

Install the prerequisite software and libraries. For MySQL you will need permission to create databases and alter user privileges, but P-SAMS itself only needs a user with SELECT privileges on P-SAMS databases.

Copy the example configuration file into the main program directory. The user custom configuration file will be ignored by git updates. Add the MySQL hostname, username and password for the P-SAMS user.

cp ./include/example.psams.conf ./psams.conf

Create a species database for P-SAMS

The following example uses specific files from http://phytozome.jgi.doe.gov, but any plant species can be added in principle.

1. From http://phytozome.jgi.doe.gov/ download the [species].transcript.fa.gz and [species].annotation_info.txt files for the desired species.
2. Unzip the FASTA file (gunzip [species].transcript.fa.gz).
3. Index the FASTA file with samtools (samtools faidx [species].transcript.fa).
4. Use the built-in script phytozome2psams.pl to process the Phytozome files (see the program instructions below).
5. Use the built-in script add_species.pl to create a kmer file from the FASTA file (see the program instructions below).
6. Create the MySQL database.
7. Add the FASTA file path and the MySQL species database name to psams.conf.

# Log into MySQL
# Create an empty database
CREATE DATABASE psams_[species];

# Grant select permissions to the P-SAMS user. Add additional security as necessary.
GRANT SELECT ON psams_[species].* TO 'psams_user'@'%';

# Use the mysql command-line tool to inialize the empty database with the P-SAMS schema
mysql -u admin_user -p psams_[species] < ./p-sams/include/structure.mysql

# Switch to the new database
USE psams_[species];

# Load the kmer file
LOAD DATA INFILE 'kmers.tab' INTO TABLE kmers FIELDS TERMINATED BY '\t' LINES TERMINATED BY '\n';

# Load the annotation file
LOAD DATA INFILE '[species].annotation.txt' INTO TABLE annotation FIELDS TERMINATED BY '\t' LINES TERMINATED BY '\n';

psams.pl

usage: psams.pl [-f FASTA] [-a ACCESSIONS -s SPECIES] [-t FOLDBACK] [-c CONSTRUCT] [-o] [-u] [-h]

Plant Small RNA Maker Suite (P-SAMS).
  Artificial microRNA and synthetic trans-acting siRNA designer tool.

arguments:
  -t FOLDBACK           Foldback type [eudicot, monocot]. Default = eudicot.
  -f FASTA              FASTA-formatted sequence. Not used if -a is set.
  -a ACCESSION          Gene accession(s). Comma-separated list. Not used if -f is set.
  -s SPECIES            Species. Required if -a is set.
  -c CONSTRUCT          Construct type (amiRNA, syntasiRNA). Default = amiRNA.
  -o                    Predict off-target transcripts? Filters guide sequences to minimize/eliminate off-targets.
  -u                    Unlimited results (slow).
  -h                    Show this help message and exit.

amiRNA_oligoDesigner.pl

usage: amiRNA_oligoDesigner.pl -s SEQUENCE -n NAME -t TYPE [-h]

Plant Small RNA Maker Suite (P-SAMS).
  Artificial microRNA oligo designer tool.

arguments:
  -s SEQUENCE           Artificial microRNA sequence. Must be 21-nucleotides long.
  -n NAME               Artificial microRNA name.
  -t TYPE               Foldback type. Options = eudicot or monocot.
  -h                    Show this help message and exit.

syntasiRNA_oligoDesigner.pl

usage: syntasiRNA_oligoDesigner.pl -s SEQUENCES -n NAMES [-h]

Plant Small RNA Maker Suite (P-SAMS).
  Synthetic trans-acting siRNA oligo designer tool.

arguments:
  -s SEQUENCES           Syn-tasiRNA sequences. A comma-separated list of one or more 21-nucleotides long sequences.
  -n NAMES               A comma-separated list of names for the syn-tasiRNA sequences. Length must match the sequences list.
  -h                     Show this help message and exit.

phytozome2psams.pl

usage: phytozome2psams.pl -f FASTA -a ANNOTATION -s SPECIES -v VERSION [-h]

Plant Small RNA Maker Suite (P-SAMS).
  Parse Phytozome transcript FASTA and annotation files.

arguments:
  -f FASTA              FASTA-formatted transcript file (*.transcript.fa).
  -a ANNOTATION         Gene annotation file (*.annotation_info.txt).
  -s SPECIES            Species name. No spaces, will be used in output file names.
  -v VERSION            Species annotation/assembly version. Will be used in output file names.
  -h                    Show this help message and exit.

add_species.pl

usage: add_species.pl -f FASTA -s SPECIES -k KSIZE [-h]

Plant Small RNA Maker Suite (P-SAMS).
  Adds a new species to the kmer database.

arguments:
  -f FASTA              FASTA-formatted transcript sequences.
  -s SPECIES            Species code.
  -k KSIZE              Kmer length.
  -h                    Show this help message and exit.