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Confirmation of a clinical diagnosis of a heritable disorder usually involves the analysis of the coding exons of a gene, plus surrounding intronic sequences. Variants that are identified using this strategy can be classed as either disease-causing or non-pathogenic polymorphisms. The variants comprise point mutations, micro insertions and deletions (indels), and copy number changes. The detection of small variants (such as substitutions and indels) can be identified using a number of techniques including SSCP (Orita et al., 1989), DHPLC (Yu et al., 2006) and highresolution melt-curve analysis (Garritano et al., 2009; Montgomery et al., 2010; NgyyenDumont et al., 2009). These techniques can de difficult to optimise and can result in both false negative and false positive results. The accepted gold standard of mutation detection, however, is dideoxy-based sequencing. This is the most direct approach and can be the most sensitive of all techniques. However, it is also the most expensive option and can be the most time-consuming due to bottle-necks arising from designing appropriate primers, the analysis of subsequent sequence data, and the interrogation of data to filter out sequence variants that are not disease-causing. Within our laboratory, we experienced difficulties in three principal areas: standardising the primer design; objectively evaluating sequence data that can be performed in an automated manner thereby addressing the bottle-neck of trace sequence evaluation; and responding to the increasing need for in-depth analysis of sequence variants to inform referring physicians as to the clinical significance of the data. To resolve the above issues, we developed a system that addressed each of these areas. The first involved designing primers that are assessed for unique amplification of targeted genomic regions using standard conditions. The aim here was not to tailor PCR conditions to a primer pair, but to force all primer designs to satisfy only one condition. The ancillary design feature of this module was to tail all primers such that all amplicons could be sequenced bi-directionally using only two primers (the “tail” sequences), thereby avoiding sequencing using amplicon-specific primers. The second area involved the wholesale adoption of Variant ReporterTM software (Applied Biosystems Ltd) for the automated analysis of all sequence data. This decision led to a significant initial investment in time to establish appropriate reference sequence projects for each of the genes analysed by our

Primer Design to Sequence Analysis - a Pipeline for a Molecular Genetic Diagnostic Laboratory