Frank M. You, Humphrey Wanjugi, Naxin Huo, Gerard R. Lazo, Ming-Cheng Luo, Olin D. Anderson, Jan Dvorak and Yong Q. Gu. RJPrimers: unique transposable element insertion junction discovery and PCR primer design for marker development. Nucleic Acids Research, 2010, Web Server Issue. doi:10.1093/nar/gkq425

Transposable elements (TE) are randomly mobilized repetitive DNA sequences in the genomes of nearly all eukaryotes. Their rapid amplification through a "cut-and-paste" or a "copy-and-paste" mechanism causes random insertion of TEs into other repetitive sequences, gene loci or intergenic regions, resulting in different types of TE or repeat junctions. Most of those junctions are unique and genome-specific, and randomly distributed along chromosomes. Thus, the TE-based junctions are a useful tool for genome-wide marker development in large and repetitive genomes, like wheat. Several PCR-based molecular marker systems based upon TEs have been proposed and applied to genetic diversity assess, and genetic and physical mapping, such as retrotransposon-based insertion polymorphism (RBIP) (Flavell et al. 1998), inter-retrotransposon amplified polymorphism (IRAP)(Kalendar and Schulman, 2006), repeat junction marker (RJM) (Wanjugi et al.), repeat junction-junction marker (RJJM) (Luce et al. 2006) and insertion-site-basedpolymorphism (ISBP)(Devos et al. 2005, Paux et al. 2006). All those marker systems need to first identify repeat junctions. Although RepeatMasker is the best known program for database based repeat screening and masking, it is not designed to identify precise repeat junctions. We often found that the junctions masked using RepeatMasker are not necessarily true junctions. To date, no high-throughput software tool is available for both repeat junction detection and fully automatic repeat junction based primer design.

RJPrimers is a high-throughput software tool to identify unique repeat junction and design TE-based primers for high-throughput marker development. This tool first identifies potentially unique repeat junctions using BLAST against fully annotated repeat databases and a repeat junction finding algorithm, and then designs TE based primers using Primer3 and BatchPrimer3. Five primer design strategies of TE based PCR markers have been implemented in this tool, including RJM, RJJM, ISBP, RBIP, and IRAP. Both a web-based server and a command line based pipeline have been implemented to meet different requirements. RJPrimers takes sequences in FASTA format as input and generates several pages of primer design results, including an HTML table page and a tab-delimited text file listing all designed primers and primer properties, and a detailed primer view page for each sequence with successfully designed primers.

Primer design strategies

• Repeat junction marker (RJM)
• Repeat junction-junction marker (RJJM)
• insertion-site-basedpolymorphism (ISBP)
• inter-retrotransposon amplified polymorphism (IRAP)
• retrotransposon-based insertion polymorphism (RBIP)

Repeat databases available for RJPrimers v1.0

• RepBase14.07 (11670 sequences)
• MIPS REdat v4.3 (5148 sequences)
• TREP complete (1503 sequences)
• Maize transposable element database (maize TEDB) (1313 sequences)
• TIGR Gramineae Repeats v2.0 (2942 sequences)
• TIGR Triticum Repeats v3.0 (452 sequences)
• TIGR Oryza Repeats v3.3 (21807 sequences)
• TIGR Hordeum Repeats v3.0 (630 sequences)
• TIGR Sorghum Repeats v3.0 (120 sequences)
• TIGR Brassica Repeats v2.0 (94 sequences)
• TIGR Brassicaceae Repeats v2.0 (224 sequences)
• TIGR Fabaceae Repeats v2.0 (28 sequences)
• TIGR Glycine Repeats v2.0 (19 sequences)
• TIGR Medicago Repeats v2.0 (23 sequences)
• TIGR Solanaceae Repeats v3.2 (181 sequences)
• TIGR Solanum Repeats v3.2 (4531 sequences)
• TIGR Arabidopsis_Repeats (250 sequences)

All those recompiled databases are downloadable at repeat_libs.tar.gz.

Program input

• Short sequences (BAC ends, shotgun sequences, Roche 454 reads or contigs, etc.) in FASTA format.
• A copy and paste to the sequence box in the RJPrimers interface.
• Upload a sequence file in FASTA format.
• The maximum of 200 short sequences (454/Sanger BAC end or any genomic sequences)in the RJPrimers web server.
• Unlimited number of sequences in the command line based pipeline of RJPrimers.

Program output

• A main HTML page containing the primer design summary of all input sequences.
• An HTML table page and a tab-delimited text file listing all designed primers and primer properties.
• A detailed primer view page for each sequence with successfully designed primers.

The primer list can be directly saved as a text file or an Excel file for further editing or primer ordering. All those pages can be downloaded as a "zip" compressed file.

Citation

Please cite the following paper if you use RJPrimers