Affordable and accesible rolony template preparation for next-generation sequencing
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The first draft of the entire human genome was released in 2000, bringing with it the potential for personalized medicine in which there would be customization of health care, with practices and decisions being specially suited to each individual patient by the use of their genetic code. However, the costs and duration of the sequencing with the available technology at that time still left genome analysis out of reach for the majority of people. Since then, there has been an ongoing challenge to lower the cost of sequencing, and to make it more accessible to the public. Newer methods of genome sequencing using circularized human DNA have now been developed that have the potential to both lower the cost and speed up the process. One such method is rolony technology in which the DNA is circularized, amplified, and then fluorescent probes are ligated to the DNA template for sequencing. The order of bases is determined by fluorescence of the ligated and bound probes. The main hurdle with this technology remains the lack of good quality sequencing templates. A good template allows for a rolony to be produced that is efficient in circularization and amplification. It has been proposed that sequence and secondary structure contribute to the quality of rolony, but the exact parameters have not yet been determined. In the work describe here, different rolony templates were chosen and studied for their sequencing potential. The hypotheses tested were whether a sequence specific secondary structure was required for circularization, whether a sequence specific secondary structure was required for Rolling Circle Amplification, and if the secondary structures assisted in folding the DNA into rolonies. It was determined through various experiments that template sequence, and the secondary structure of the template are representative of the quality of rolony produced.
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