Establishing a canine genome sequencing protocol using Oxford Nanopore

Sammanfattning: The development of sequencing technologies has led to monumental ad-vances in the field of genomics, creating new areas of investigation and profoundly impacting our understanding of life itself. Presently, the “third generation” of these technologies is focused on improving the sequencing of long reads, which allows for studying complex areas in the genome. A promising platform offering long-read sequencing at a comparatively low cost is the Oxford Nanopore Technologies “MinION,” a USB-connected device the size of an ordinary dongle, which can be used in as good as any laboratory setting with a consumer-grade computer. Given that the techno-logy is both recent and still under development, however, there is a need to formulate and verify adequate methodologies for a great variety of target species. In this thesis, a protocol for long-read sequencing of canine DNA using the MinION is presented. Four different HMW-gDNA extraction methods and five library preparation variants were evaluated in order to determine which approach would generate the best sequencing results. Additionally, a method for reusing flow cells in order to maximize data ge-nerated per cell and reducing costs was tested and deemed successful. Major challenges encountered throughout the project include DNA quality, fragment length, as well as high rates of pore loss and low pore occu-pancy. The best-performing DNA extraction protocol was an altered version of Qiagen's Genomic-tip 100/G. For library preparation, a modified version of Nanopore's Sequencing by Ligation kit (SQK-LSK109) had the most favourable results. The best sequencing run generated 14 Gbp of raw data in the span of 48 hours. The results presented herein constitute a first step towards the establishment of a method that leverages the MinION's advan-tages in canine genome sequencing projects.