Optimising probe library design for DNA resequencing

Haslam, Niall (2007) Optimising probe library design for DNA resequencing. University of Southampton, Doctoral Thesis.

Abstract

This work focuses on the development of a set of analysis tools for the design of sequencing by hybridisation experiments. Sequencing by hybridisation is an effective method for obtaining large amounts of DNA sequence information at relatively low cost. It is well suited to applications such as resequencing where the target sequence is already known and the experiment is designed to detect known polymorphisms. Sequencing by hybridisation uses short oligonucleotide probes to indirectly detect the presence of a complementary sequence in the target sequence. The optimisation of this process reflects the requirements to create unique probes, with similar thermal properties, that will not cross-hybridise and are capable of providing coverage for as much of the target as possible. Reducing the probe length can bring significant savings to the method and drastically reduce the costs of a sequencing by hybridisation experiment. The complex interplay between target sequence variability, number of reference sequences used, thermal filtering, likelihood of cross-hybridisation and optimal probe length is investigated. The results of this examination of a number of targets are shown to demonstrate the power of the analysis in making decisions about the choice of resequencing method. Using the analysis tools developed during this work a number of predictions are made about the optimal probe length for resequencing the example targets. The design focuses on resequencing these targets using reference sequences to probe for previously observed sequence variants. The results show that the optimal probe length and number of reference sequences used are target dependent. This suggests the need to develop and apply a general purpose tool for the design of resequencing libraries.

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