BACKGROUND: Next generation sequencing technologies often require numerous primer designs that require good target coverage that can be financially costly. We aimed to develop a system that would implement primer reuse to design degenerate primers that could be designed around SNPs, thus find the fewest necessary primers and the lowest cost whilst maintaining an acceptable coverage and provide a cost effective solution. We have implemented Metropolis-Hastings Markov Chain Monte Carlo for optimizing primer reuse. We call it the Markov Chain Monte Carlo Optimized Degenerate Primer Reuse (MCMC-ODPR) algorithm. RESULTS: After repeating the program 1020 times to assess the variance, an average of 17.14% fewer primers were found to be necessary using MCMC-ODPR for an equivalent coverage without implementing primer reuse. The algorithm was able to reuse primers up to five times. We compared MCMC-ODPR with single sequence primer design programs Primer3 and Primer-BLAST and achieved a lower primer cost per amplicon base covered of 0.21 and 0.19 and 0.18 primer nucleotides on three separate gene sequences, respectively. With multiple sequences, MCMC-ODPR achieved a lower cost per base covered of 0.19 than programs BatchPrimer3 and PAMPS, which achieved 0.25 and 0.64 primer nucleotides, respectively. CONCLUSIONS: MCMC-ODPR is a useful tool for designing primers at various melting temperatures at good target coverage. By combining degeneracy with optimal primer reuse the user may increase coverage of sequences amplified by the designed primers at significantly lower costs. Our analyses showed that overall MCMC-ODPR outperformed the other primer-design programs in our study in terms of cost per covered base.

SUMMARY: TreeMos is a novel high-throughput graphical analysis application that allows the user to search for phylogenetic mosaicism among one or more DNA or protein sequence multiple alignments and additional unaligned sequences. TreeMos uses a sliding window and local alignment algorithm to identify the nearest neighbour of each sequence segment, and visualizes instances of sequence segments whose nearest neighbour is anomalous to that identified using the global alignment. Data sets can include whole genome sequences allowing phylogenomic analyses in which mosaicism may be attributed to recombination between any two points in the genome. TreeMos can be run from the command line, or within a web browser allowing the relationships between taxa to be explored by drill-through. AVAILABILITY: http://www2.warwick.ac.uk/fac/sci/whri/research/archaeobotany.