Biomolecular Detection and Quantification · 2015 · 597 citations · 31 references
Life SciencesMicrobiologyMolecular BiologyGenome AssemblyGenomicsMicrobial Genomics16S Ribosomal Ribonucleic Acid AnalysisDna SequencingGenome SequencingNext-generation SequencingError Rate AnalysisError Rate MetricsNanopore Sequencing
The Oxford Nanopore Technologies MinION is a low‑cost, portable, real‑time sequencer capable of producing long reads that could improve genome assemblies and resolve repeat‑rich regions, but its performance and accuracy must be evaluated before widespread adoption. The study aimed to assess the MinION’s performance. This was done by re‑sequencing three bacterial genomes with diverse GC content, noting underrepresentation of the high‑GC strain. The MinION’s base‑called error rate was 38.2 %, with mean and median read lengths of 2 kb and 1 kb, a longest read of 98 kb, and a single read spanning a 5 kb rRNA operon, yet its high error rate limits competitiveness, though it can improve de novo assembly contiguity when combined with Illumina MiSeq data.
The Oxford Nanopore Technologies (ONT) MinION is a new sequencing technology that potentially offers read lengths of tens of kilobases (kb) limited only by the length of DNA molecules presented to it. The device has a low capital cost, is by far the most portable DNA sequencer available, and can produce data in real-time. It has numerous prospective applications including improving genome sequence assemblies and resolution of repeat-rich regions. Before such a technology is widely adopted, it is important to assess its performance and limitations in respect of throughput and accuracy. In this study we assessed the performance of the MinION by re-sequencing three bacterial genomes, with very different nucleotide compositions ranging from 28.6% to 70.7%; the high G + C strain was underrepresented in the sequencing reads. We estimate the error rate of the MinION (after base calling) to be 38.2%. Mean and median read lengths were 2 kb and 1 kb respectively, while the longest single read was 98 kb. The whole length of a 5 kb rRNA operon was covered by a single read. As the first nanopore-based single molecule sequencer available to researchers, the MinION is an exciting prospect; however, the current error rate limits its ability to compete with existing sequencing technologies, though we do show that MinION sequence reads can enhance contiguity of de novo assembly when used in conjunction with Illumina MiSeq data.
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