Flexible and Scalable Automation Solutions For Scoring Single Nucleotide Polymorphisms Using Rolling Circle Amplification

A single nucleotide polymorphism (SNP) scoring assay that uses ligation-dependent Rolling Circle Amplification (RCA)† was transferred to a series of automated protocols addressing a range of throughput levels. The systems utilised various automation modules consisting of custom-made and offthe-shelf devices. Several system parameters were evaluated to ensure assay integrity and homogeneity. These included reagent carry over, liquid evaporation rates, thermal regulation of reactions and fluorescence reading capabilities.Data analysis software was developed in order to rapidly allocate SNP calls from data generated by the automated system. A modified fuzzy c -means clustering algorithm was employed to separate data points into groups associated with specific genotypes. Data were then presented graphically and within a summary table, which allowed easy and rapid organization and interpretation of data.