Croning et al. BMC Genomics 2010, 11:74 http://www.biomedcentral.com/1471-2164/11/74 Page 4 of 7 and failed groups. Redundant (but passed) sequences are filtered into a fourth bin. If none of the sequences in the probe design pass at the specified criteria, the cut-offs are automati- cally relaxed to find the best (but poorly-scoring) probes in what is likely to be a difficult portion of the genome to design Southern blot probes. Pri- mer3 [12] is then used to generate primers for recovery of the passed candidate probes, run using the BioPerl-Run wrapper [13]. Chosen primers can be manually-checked for potentially confounding polymorphisms if required, by search of dbSNP. Static web output is generated for user inspection (see Figure 2). This includes the probe design win- dow coordinates, counts of the candidate probes generated and subsequently placed in each bin and results of the quality assurance checks that each genome search generates an ‘on target’ hit to the correct position on the genome for the candidate probe sequence. A graphic plot is rendered showing the frequency of occurrence of each base position in the set of sequences found in the unique, passed or failed probe groups across the design window. Four accessory scripts are also provided. create_pro- be_search_db_tables creates the MySQL database tables for the design pipeline. delete_probe_design removes a probe design from the database should it have been wrongly specified, or is no longer needed. delete_job_re- sults removes the results of a particular batch of Exoner- ate genome searches from the database should an error have occurred, allowing the job to be resubmitted, and finally, get_probe_search_cpu_time calculates the total time to execute the searches for a given probe design. Figure 2 Screenshot of final web output following a completed Southern blot probe design, search and analysis run. In this example a probe is designed against a 3 kb window on chromosome 2 of the NCBI37 mouse genomic assembly. 458 candidate probes were tested and many unique probes were found, 301/458 (shown in yellow). The remainder (157/458) passed the empirical cut-off criteria (shown in green) suggesting this region of the chromosome is likely composed of quite unique sequence, almost free from repetitive and low-complexity DNA elements.