![]() The MinION uses small consumable flowcells for sequencing, which contain fluid channels that flow samples onto a sequencing matrix and provide a small amount of fluid waste storage. It has very low capital cost, has the potential to generate more than 1 Gb of sequence data per 100 USD, has a footprint about the size of an office stapler and runs on a standard desktop or laptop computer. ![]() ![]() The Oxford Nanopore Technologies (ONT) MinION makes long-read sequencing accessible to most laboratories outside of a dedicated genome facility. In short, nanopore sequencing solves the technical challenges of reading long DNA fragments, while still having room for improvement in terms of per read accuracy. The number of genome assemblies using nanopore data either exclusively or in combination with other sequencing data is steadily increasing, for example the 3.5 gigabase (Gb) human genome, the 860 Mb European eel genome, the 1 Gb genome of the wild tomato species Solanum pennellii and the 135 Mb genome of Arabidopsis thaliana (Jain et al., 2018 Jansen et al., 2017 Michael et al., 2018 Schmidt et al., 2017). Long reads have a number of important applications, including improving the accuracy and efficiency of genome assembly, especially for genomes that contain long low-complexity regions detailed investigation of segmental duplications and structural variation (Jain et al., 2018) major histocompatibility complex (MHC) typing (Liu et al., 2017) and detecting methylation patterns (Simpson et al., 2017). This technology can sequence DNA fragments of varied lengths, from a few hundred bases to over a megabase (Mb), which compares favourably to sequencing by synthesis (e.g., Illumina), which is limited to hundreds of bases (Leggett & Clark, 2017). Groups of consecutive bases cause a characteristic shift in current, and this can be deconvoluted to infer the individual base sequence of the DNA molecule, a process referred to as basecalling. Single-molecule nanopore sequencing records changes in electrical current as individual tagged DNA molecules pass through an engineered pore across a chemical gradient (Jain, Olsen, Paten, & Akeson, 2016). We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls and suggestions of how to adapt it to other tissue types, will be useful to others who plan to establish long-read sequencing in their own laboratories. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. Our protocols are open source and can be performed in any laboratory without special equipment. ![]() Following the workflow illustrated, we were able to reliably and repeatedly obtain >6.5 Gb of long-read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Here, we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimizing DNA extraction from a challenging plant tissue as a case study. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time-consuming and costly. This is especially the case for the portable MinION sequencer which enables all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. Realizing their full potential is critically reliant on extracting high-quality, high-molecular-weight (HMW) DNA from the organisms of interest. Long-read sequencing technologies are transforming our ability to assemble highly complex genomes.
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