Browsing by Author "Koivunen, Sampo"

The Oxford Nanopore MinION is a third generation sequencer utilizing nanopore sequencing technology. The nanopore sequencing method allows sequencing of either DNA or RNA strands as they pass through the membrane-embedded nanopores. By measuring the corresponding fluctuations in the ion flow passing through the nanopore the passing strands can be sequenced directly without additional second-hand reactions or measurements. The MinION sequencing has very distinctly different characteristics compared to the market leaders of the sequencing field. The small form factor of the device further helps it to separate itself from the other alternatives. However, the technology has only been on the market for a very short time and thus very little golden standards regarding its capabilities or usage have been established. This thesis describes our experiences testing the capabilities of the MinION sequencer both before its commercial release as a part of a special early access program, as well as our continued experiments with the device following its commercial launch. The main results of this study include successfully sequencing and aligning E.coli and human gDNA samples to their respective reference genomes. Using our sequencing and analysis pipeline specifically tuned to the MinION we were able to sequence the entire E.coli genome on a single MinION flow cell with an average depth of around 180. Over the course of the thesis project the MinION sequencing protocol was evaluated and optimized in order to determine whether it has the potential to achieve our ultimate goal of reliably sequencing the previously inaccessible genomic regions of the human genome. The possibility of augmenting the sequencing protocol by adding the pre-sequencing target enrichment was also explored. Ultimately we were able to confirm that the MinION sequencer can be used to sequence long DNA fragments from a multitude of sample types. The majority of the produced reads could successfully be aligned against a reference genome. However, the limited yield and sequencing quality of a single experiment does limit the applicability of the method for more complicated genomic studies. These issues can be addressed with various techniques, chiefly target enrichment, but adapting such methods into the sequencing pipeline has its own challenges.