Primo Oligo Design Code
To complement the MerMade systems are a series of informatics tools to accurately design primers for PCR and for sequencing dye terminator reactions. The heart of this system is a new code, PRIMO, which does primer design based on a quality assessment of the template sequence from which the primers are being designed. This code avoids regions of questionable sequence quality by analyzing automated sequence output ‘trace files’ directly or by inputting quality data from the codes, phred and phrap (assembly code produced by Phil Green, University of Washington, Seattle) used in sequence assembly, thus making oligos with a higher probability of functioning well after manufacture.
PRIMO is a computer program that designs walking primers for large-scale DNA sequencing projects. Oligonucleotide primers are predicted automatically, using quality information associated with each base call, eliminating the need for manually viewing the sequence traces or inspecting contig assemblies to determine appropriate locations for primer design. This allows PRIMO to run in batch mode on an arbitrarily large number of templates. For shotgun sequencing, PRIMO reads assembled sequence contigs with corresponding base quality statistics and automatically designs walking primers as needed to extend and join contigs, or improve their overall quality. In the opposite extreme of single pass or completely directed sequencing, PRIMO reads the unassembled sequence for each template and designs walking primers for extending each read. If the base calling software does not provide base quality statistics, PRIMO assigns its own measure of base quality determined by the shapes of individual peaks in the trace data for each template. In this way, PRIMO can either be used in the finishing stages of a shotgun sequencing project, in sequencing by directed primer walking, or in some intermediate strategy. The code is written in ANSI C and maintained in two versions: one for the Macintosh and the other for UNIX.
For example, for sequence walking, for primers designed by PRIMO W.1 there is general agreement between the primer location and the typical positioning of the high quality sequence regions within the overall read. As usual the high quality regions lie away from the extreme ends of the read. Balanced with the desire to predict forward and reverse primers in good sequence regions (away from the ends of the read) is the desire to predict primers as near as possible to the ends of the good sequence region so that the read generated from the primer will contribute as much as possible towards extending the original sequence.
