Fig. 1

PrimerXL targeted resequencing PCR design pipeline. Schematic representation of the primerXL pipeline functionality. Tasks performed by the parent process are marked in yellow, tasks executed by the child processes in green and blue. The design criteria relaxation cascade is shown in blue. Following retrieval of the sequence of interest (and adjustment of the amplicon size range for small regions), SNPs and secondary structures are masked. Next Primer3-based primer design is initiated and a SNP- and secondary structure analysis is performed on each resulting assay to remove assays harboring these features in their primer annealing sites. If a successful primer pair remains after specificity assessment, the next target region is processed or the design is terminated. On the other hand, if no successful assays remain, design parameters are relaxed (specificity-, SNP- and secondary structure analysis stringency, GC content, amplicon length, …) and primer design is attempted once again. If all parameters have already been relaxed to their fullest extent without the generation of a successful primer pair, the design process is terminated