Nanopore MinION sequencing

The Oxford Nanopore MinION DNA sequencing system uses a novel 'nanopore technology for sequencing long streches of DNA. Briefly, large nanopores are inserted into a systhetic, electrically resistant membrane. A voltage is set across the membrane establishing an ionic current across the pores.  DNA molecules tagged with tether proteins are guided to nanopores and the electric field drives individual DNA strands through biological nanopores at a rate  controlled by a processive enzyme bound to the DNA at the pore orificeby. Each base ia identified by measuring the changes in current across the nanopores. Read length reflects the length of the DNA fragments submitted for sequencing.
More information about this technology can be found at https://nanoporetech.com.

MinION long reads are being used to close gaps and join contigs obtained from assemblies of Illumina reads of small genomes or genomic loci and the MCIC Computational Biology Laboratory has the necessary bioinformatics tools set up.


Library preparation and sequencing

Library preparation entails a DNA fragmentation step, ligation of the adaptors (with or without indices), optional PCR amplification and final attachment of the tether/motor protein to the 5' end of the DNA molecules. Input DNA can be as low as 20ng, however, for a PCR free library preparation protocol at least 1-2 ug of DNA are needed. Preparation of intact, not nicked high molecular weight genomics DNA is crucial for obtaining long and good quality reads.

Sequencing takes approximately 48 hours. During the run data are collected locally and uploaded to the Nanopre cloud computing where they are analysed using the Metrichor software. Metrichore software also provides real time feedback on the quality of the run. The lenght of the reads depends on the fragmentation size of the DNA and on its quality (no nicks). The system is capable of producing sequence reads of more than 200Kb and the thoughput for a flowcell is currently ~100-200 Mb. Several different templates can be sequenced sequencially on a flow cell if a lower throughput is needed for each individual template.