Scaffolding of a bacterial genome using MinION nanopore sequencing (original) (raw)
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The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community
Genome Biology, 2016
Nanopore DNA strand sequencing has emerged as a competitive, portable technology. Reads exceeding 150 kilobases have been achieved, as have in-field detection and analysis of clinical pathogens. We summarize key technical features of the Oxford Nanopore MinION, the dominant platform currently available. We then discuss pioneering applications executed by the genomics community.
Genome assembly using Nanopore-guided long and error-free DNA reads
BMC Genomics, 2015
Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments.
Assessing the performance of the Oxford Nanopore Technologies MinION
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...
BMC Genomics
Background: Short-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated. Results: We sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these assemblies were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions. Conclusions: Genome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes with sufficient accuracy to enable the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing can therefore costeffectively advance studies of microbial evolution and genome-driven drug discovery.
Nanopore sequencing data analysis: state of the art, applications and challenges
The nanopore sequencing process is based on the transit of a DNA molecule through a nanoscopic pore, and since the 90s is considered as one of the most promising approaches to detect polymeric molecules. In 2014, Oxford Nanopore Technologies (ONT) launched a beta-testing program that supplied the scientific community with the first prototype of a nanopore se-quencer: the MinION. Thanks to this program, several research groups had the opportunity to evaluate the performance of this novel instrument and develop novel computational approaches for analyzing this new generation of data. Despite the short period of time from the release of the MinION, a large number of algorithms and tools have been developed for base calling, data handling, read mapping, de novo assembly and variant discovery. Here, we face the main computational challenges related to the analysis of nanopore data, and we carry out a comprehensive and up-to-date survey of the algorithmic solutions adopted by the bioinformatic community comparing performance and reporting limits and advantages of using this new generation of sequences for genomic analyses. Our analyses demonstrate that the use of nanopore data dramatically improves the de novo assembly of genomes and allows for the exploration of structural variants with an unprecedented accuracy and resolution. However, despite the impressive improvements reached by ONT in the past 2 years, the use of these data for small-variant calling is still challenging, and at present, it needs to be coupled with complementary short sequences for mitigating the intrinsic biases of nanopore sequencing technology.
NanoPipe - a web server for nanopore MinION sequencing data analysis
GigaScience
Background: The fast-moving progress of the third-generation long-read sequencing technologies will soon bring the biological and medical sciences to a new era of research. Altogether, the technique and experimental procedures are becoming more straightforward and available to biologists from diverse fields, even without any profound experience in DNA sequencing. Thus, the introduction of the MinION device by Oxford Nanopore Technologies promises to "bring sequencing technology to the masses" and also allows quick and operative analysis in field studies. However, the convenience of this sequencing technology dramatically contrasts with the available analysis tools, which may significantly reduce enthusiasm of a "regular" user. To really bring the sequencing technology to every biologist, we need a set of user-friendly tools that can perform a powerful analysis in an automatic manner. Findings: NanoPipe was developed in consideration of the specifics of the MinION sequencing technologies, providing accordingly adjusted alignment parameters. The range of the target species/sequences for the alignment is not limited, and the descriptive usage page of NanoPipe helps a user to succeed with NanoPipe analysis. The results contain alignment statistics, consensus sequence, polymorphisms data, and visualization of the alignment. Several test cases are used to demonstrate the efficiency of the tool. Conclusions: Freely available NanoPipe software allows effortless and reliable analysis of MinION sequencing data for experienced bioinformaticians, as well for wet-lab biologists with minimum bioinformatics knowledge. Moreover, for the latter group, we describe the basic algorithm necessary for MinION sequencing analysis from the first to last step.
MinION Nanopore Sequencing Accelerates Progress towards Ubiquitous Genetics in Water Research
Water
In 2014, Oxford Nanopore Technologies (ONT) introduced an affordable and portable sequencer called MinION. We reviewed emerging applications in water research and assessed progress made with this platform towards ubiquitous genetics. With >99% savings in upfront costs as compared to conventional platforms, the MinION put sequencing capacity into the hands of many researchers and enabled novel applications with diverse remits, including in countries without universal access to safe water and sanitation. However, to realize the MinION’s fabled portability, all the auxiliary equipment items for biomass concentration, genetic material extraction, cleanup, quantification, and sequencing library preparation also need to be lightweight and affordable. Only a few studies demonstrated fully portable workflows by using the MinION onboard a diving vessel, an oceanographic research ship, and at sewage treatment works. Lower nanopore sequencing read accuracy as compared to alternative platfor...
Genomics, 2021
Oxford Nanopore sequencing has been widely used to achieve complete genomes of bacterial pathogens. However, the error rates of Oxford Nanopore long reads are high. Various polishing algorithms using Illumina short reads to correct the errors in Oxford Nanopore long-read assemblies have been developed. The impact of polishing the Oxford Nanopore long-read assemblies of bacterial pathogens with Illumina short reads on improving genomic analyses was evaluated using both simulated and real reads. Ten species (10 strains) were selected for simulated reads, while real reads were tested on 11 species (11 strains). Oxford Nanopore long reads were assembled with Unicycler to produce a draft assembly, followed by three rounds of polishing with Illumina short reads using two polishing tools, Pilon and NextPolish. One round of NextPolish polishing generated genome completeness and accuracy parameters similar to the reference genomes, whereas two or three rounds of Pilon polishing were needed, though contiguity remained unchanged after polishing. The polished assemblies of Escherichia coli O157:H7, Salmonella Typhimurium, and Cronobacter sakazakii with simulated reads did not provide accurate plasmid identifications. One round of NextPolish polishing was needed for accurately identifying plasmids in Staphylococcus aureus and E. coli O26:H11 with real reads, whereas one and two rounds of Pilon polishing were necessary for these two strains, respectively. Polishing failed to provide an accurate antimicrobial resistance (AMR) genotype for S. aureus with real reads. One round of polishing recovered an accurate AMR genotype for Klebsiella pneumoniae with real reads. The reference genome and draft assembly of Citrobacter braakii with real reads differed, which carried blaCMY-83 and fosA6, respectively, while both genes were present after one round of polishing. However, polishing did not improve the assembly of E. coli O26:H11 with real reads to achieve numbers of virulence genes similar to the reference genome. The draft and polished assemblies showed a phylogenetic tree topology comparable with the reference genomes. For multilocus sequence typing and pan-genome analyses, one round of NextPolish polishing was sufficient to obtain accurate results, while two or three rounds of Pilon polishing were needed. Overall, NextPolish outperformed Pilon for polishing the Oxford Nanopore long-read assemblies of bacterial pathogens, though both polishing strategies improved genomic analyses compared to the draft assemblies.
Improved data analysis for the MinION nanopore sequencer
Nature Methods, 2015
Speed, single-base sensitivity and long read lengths make nanopores a promising technology for high-throughput sequencing. We evaluated and optimized the performance of the MinION nanopore sequencer using M13 genomic DNA and used expectation maximization to obtain robust maximum-likelihood estimates for insertion, deletion and substitution error rates (4.9%, 7.8% and 5.1%, respectively). Over 99% of high-quality 2D MinION reads mapped to the reference at a mean identity of 85%. We present a single-nucleotide-variant detection tool that uses maximum-likelihood parameter estimates and marginalization over many possible read alignments to achieve precision and recall of up to 99%. By pairing our high-confidence alignment strategy with long MinION reads, we resolved the copy number for a cancer-testis gene family (CT47) within an unresolved region of human chromosome Xq24.