Abstract
Electronic, nanopore based single molecule real-time DNA sequencing technology offers very long, albeit lower accuracy reads in sharp contrast to existing next-generation sequencing methods, which offer short, high-accuracy reads in abundance. We provide a systematic review of the error characteristics of this new sequencing platform, and demonstrate the most challenging aspects in the field of whole gene sequencing through the human HLA-DQA2 gene using long-range PCR products on multiplexed samples. We consider the limitations of these errors for the applications of this technology, and also indicate prospective improvements and expected thresholds with respect to these errors.
Highlights
After decades of DNA sequencing approaches based on sequencing-by-synthesis [1], Oxford Nanopore Technologies (ONT) introduced a commercially available electronic DNA sequencing technology in 2014: the MinION portable single-molecule real-time DNA sequencing platform
In this paper we provide a systematic investigation of nanopore-based DNA sequencing errors using the MinION with R7.3 flow cells and using the HLA-DQA2 gene as a target
5 Conclusion The initial capital cost of next-generation sequencing (NGS) starts above 50k USD, while the MinION device promises capital costs under 1k USD
Summary
After decades of DNA sequencing approaches based on sequencing-by-synthesis [1], Oxford Nanopore Technologies (ONT) introduced a commercially available electronic DNA sequencing technology in 2014: the MinION portable single-molecule real-time DNA sequencing platform. Current next-generation DNA sequencing methods –except for Pacific Biosciences’ Helicos technology– use clonal clusters of identical DNA strands to perform sequencing by synthesis. In the case of Life Technology’s IonTorrent platform, template strands are amplified on immobilized beads by emulsion polymerase chain reaction The sequencing is performed by flowing alternating deoxynucleotides over a plate with a single bead in each well, and recording the pH change induced by the incorporation of nucleotides. In the current next-generation sequencing market leader Illumina’s platform, the target DNA fragments of interest are amplified in situ using bridge PCR on glass plate with immobilized probes, while the sequencing is performed by fluorescent labelled reversible terminator sequencing.
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