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Versatile sequencing library preparation methods for MinION, GridION and PromethION

A comprehensive range of gDNA and amplicon library preparation kits and protocols is available, offering high throughput, low DNA input, rapid preparation and ultra-long reads.

Fig. 1 Schematic representation of PCR-free library preparation a) ligation b) rapid

PCR-free library preparation, for low bias, long fragment native DNA sequencing

It is not necessary to perform amplification of any kind prior to nanopore sequencing. There are several advantages to this: amplification bias is eliminated; fragment lengths (and hence read lengths) are not limited by the processivity of a polymerase enzyme; PCR-free library preparation saves time, compared to PCR-based methods; methylation and other modifications are retained. We have two basic PCR-free genomic DNA library preparation approaches. The ligation-based approach (Fig. 1a) offers greater control over library fragment size, whereas the transposase-based approach (Fig. 1b) is extremely quick to perform, allowing libraries to be made in approximately 10 minutes.

Fig. 2 Low-input PCR-based library preparation a) ligation b) rapid

PCR-based library preparation for lower quantities of starting DNA

In situations where the quantity of starting genomic DNA is limited (1 ng–100 ng) we have released versions of our library preparation methods in which universal adapters are attached to the ends of DNA fragments, and PCR is used to increase the quantity of library DNA. As with the PCR-free methods, one of the low-input protocols is ligation-based and the other uses a transposase enzyme. In both approaches, PCR primers are used which have Oxford Nanopore’s rapid attachment chemistry at their 5’ end. This means that, following amplification, sequencing adapters can be attached to the ends of the amplicons in a single-step, 10-minute reaction, without the need to clean up the amplicons beforehand.

Fig. 3 a) standard whole-genome amplification b) rapid whole-genome amplification workflows

Whole-genome amplification protocols for picogram levels of input DNA

For some sample types it is not possible to begin library preparation with nanogram quantities of DNA. This is particularly true when working with single cells, or small numbers of cells. To address this need we have developed a whole-genome amplification (WGA) protocol. The first part of the process utilises a commercial WGA kit, which can amplify DNA from single cells without the need to extract DNA beforehand. In the standard protocol the hyperbranched WGA DNA is digested to remove flaps and other irregular structures, and sequencing adapters are added (Fig. 3a). In the rapid WGA protocol (Fig. 3b), the WGA reaction itself is performed for a shorter period of time, and the hyperbranched DNA is prepared for sequencing using the rapid gDNA kit (Fig. 1b)

Fig. 4 Library preparation for higher single-read accuracy a) 1D2 workflow b) 1D2 accuracy

Library preparation options for greater single-read accuracy

The single-read accuracy obtained using our standard 1D library preparation and analysis workflows is sufficient for many sequencing applications. However, there are circumstances in which it would be an advantage to have higher single-read accuracy. We have recently introduced a ‘1D2’ library preparation workflow in which an adapter is attached to DNA duplexes which allows both strands in the duplex to be sequenced consecutively, even though the two strands are not joined together (Fig. 4a). The two sets of data from the duplex are combined before basecalling to generate a single, higher-accuracy basecalled read. We generated both 1D and 1D2 datasets from E. coli and calculated the single-read accuracies (Fig. 4b). The modal 1D2 accuracy was approximately 96%.

Using long nanopore reads to assemble genomes from complex metagenomic samples

The long reads of nanopore technology allow assembly of individual genomes from complex mixtures of  different organisms, with good assembly contiguity

Fig. 1 De novo metagenomic assembly a) laboratory workflow c) bioinformatic pipeline

Long reads provide overlaps and improve genome assembly from complex samples

Because the vast majority of microbial species cannot be cultured in the laboratory, the most direct way to derive the whole genome sequences of complex mixtures of organisms is by metagenomic assembly. Here, the different genomes within the sample are sequenced and assembled together (Fig. 1a). Metagenomic mixtures typically contain a large number of similar  genomes, with vastly different levels of abundance, which can lead to misassembly. Long reads are less susceptible to misassembly than shorter reads because they give longer regions of overlap. We have developed a bioinformatic pipeline for metagenomic assembly (Fig. 1b), in which reads are first binned according to species and the bins are then assembled separately. 
 

Fig. 2 Metagenomic assembly statistics for ZymoBIOMICS’ Microbial Community DNA Standard

Validation of metagenomic assembly pipeline using microbial mock community

We used the ZymoBIOMICS Microbial Community DNA Standard to validate the pipeline described in Fig. 1. This mixture contains eight bacterial and two fungal genomes. We generated ~10 Gb of 1D FASTA data with a mean read length of ~5 kb (limited by sample DNA integrity). Summary assembly statistics for the eight bacteria are shown in Fig. 2a. Although single-contig reference assemblies of the exact strains used in the community standard were not available for comparison, we identified the closest matches in the GenBank database by BLASTing, and constructed mummer plots of our assemblies using these. In this way, we were able to confirm that our metagenomic assembly pipeline yielded trustworthy results (Figs. 2b and 2c).

Fig. 3 Metagenomic assembly of bacteria in a probiotic food supplement

Metagenomic assembly of bacterial genomes from a probiotic food supplement

To demonstrate the effectiveness of the assembly pipeline on nanopore data from a real metagenomic sample, we sequenced DNA from a probiotic food supplement which contained 15 known bacteria. We generated a total of ~10 Gb of 1D FASTA data, from a single run. Figs. 3a and 3b show assembly statistics from this dataset. We produced a total of 104 contigs, with a mean length of ~308 kb. Several of the contigs obtained were close in size to the published reference sequences for the nearest strains, as identified by BLAST. Mummer plots of two of the assembled genomes, Lactobacillus acidophilus and Streptococcus thermophilus, with the closest references, are shown in Figs. 3c and 3d respectively. The assemblies shown are perfectly contiguous and cover the entire length of the reference assemblies.  

Fig. 4 Assembly and analysis of bacteria present in soil taken from around Basmati rice roots

Assembly of bacterial genomes from a high- complexity soil sample

To test the assembly pipeline on a more complex sample, we extracted genomic DNA from soil obtained from around the roots of Basmati rice plants. We obtained approximately 4 million reads > 1,000 nt in length and with a quality score > 10. High-complexity metagenomic samples rarely provide single-contig assemblies because of the presence of many closely-related organisms, which have highly homologous genomes, and which are present over a wide range of abundance (Fig. 4a). Assembly yielded a large number of long contigs, from a wide range of bacterial and fungal species. Fig. 4b shows an example of an assembled Hyphomicrobium species from the soil sample. Although the assembly consists of several contigs, this is no impediment to performing functional characterisation of the genes present (Fig. 4c).

Metagenomic analysis of 30,000-year-old microbes from Siberian permafrost

A metagenomic survey of microbial DNA extracted from 30,000-year-old permafrost, and genome assembly of a giant virus which was resurrected from the sample

Fig. 1: Extraction of permafrost sample

Permafrost is an ideal storage medium for DNA preservation

Permafrost is a thick layer of soil, lying beneath ice or other soil, which stays frozen throughout the year. Permafrost in Chukotka, Siberia, is known to have remained frozen for tens of thousands of years (Fig. 1). Permafrost is an ideal storage medium for DNA preservation. We obtained DNA from a 30,000-year-old Siberian permafrost sample which had never thawed, and performed metagenomic analysis using our WIMP workflow. WIMP performs real-time species identification, using kraken to determine the most likely placement of a sequence in the taxonomy tree, and to give each placement a classification score. The higher this score, the higher the confidence in the classification

Fig. 2 WIMP analysis of permafrost DNA a) fungi, archaea and viruses b) bacteria

Analysis of permafrost DNA offers snapshot of life 30,000 years ago

The WIMP workflow currently analyses bacteria, viruses and fungi together, and we found DNA from examples of each type of microorganism in the sample. For the sake of clarity, results for viruses and fungi (Fig. 2a) are shown separately from those for bacteria (Fig. 2b).

Intact amoeba cells have been observed in Siberian permafrost samples. Because giant DNA viruses have been observed within modern amoeba cells, collaborators at CNRS in Marseille suspected that there could be preserved giant amoeba-infecting viral particles trapped in Siberian permafrost.

Fig. 3 ONT-only assembly of Pithovirus sibericum

30,000-year-old virus resurrected from Siberian permafrost

Our collaborators discovered a viable amphora-shaped virus in the Siberian permafrost sample (Fig. 3a). The virus was found to infect Acanthamoeba, allowing it to be cultured in the laboratory and studied. It was given the  name Pithovirus sibericum. By viral standards, Pithovirus is enormous, 1.5 mm long, and has a large AT-rich genome, approximately 600 kb in length. We prepared a low-input library from the cultured virus, and sequenced this on the MinIONTM. We assembled the genome using Nanocorrect and the Celera Assembler, obtaining a single contig of 620 kb. Our assembly agrees well with the published assembly, as illustrated in the Mummer plot (Fig. 3b).

Fig. 4 Thawing permafrost may release viable pathogens

Viable pathogens may be released from melting permafrost

Pithovirus shares several features, including genome structure and replication cycle, with other large eukaryote-infecting viruses, raising the possibility that other viruses are also preserved in permafrost in a viable state. As a consequence, melting of permafrost, as a result of climate change, mining or drilling may release these potential pathogens. No living cellular organisms have yet been definitively obtained from samples of this age, but some pathogenic bacteria are known to be capable of survival under the low temperatures encountered in circumpolar regions. It is therefore advisable to perform metagenomic surveys more widely on permafrosts to document the pathogens present. 

Incorporating sequence capture into library preparation for MinION, GridION and PromethION

Hybrid sequence capture allows users to select thousands of loci of interest simultaneously prior to sequencing, making more efficient use of the sequencing run

Fig. 1 Long-read sequence capture a) workflow overview b) multiplexed sequence capture

Sequence capture uses complementary probes to enrich for targets of interest

Sequence capture is a technique which allows the enrichment of specific regions of interest from a genome. It is useful when:
i) the user is not interested in analysing the entire genome
ii) the genome is too large for the throughput of the sequencer
iii) the user wishes to save money and time on sequencing and analysis
iv) the regions are longer than can be amplified by PCR, or too many PCRs would be required.

Sequence capture is performed during library preparation by hybridising the library fragments to probes which are specific to the regions of interest (Fig. 1).

Fig. 2 Analysis report of sequence-capture data for the human exome

Resequencing analysis workflow for sequence-capture experiments

We have released an updated analysis workflow for sequence-capture experiments. To illustrate this, we captured the human exome using Agilent’s SureSelect Human All Exon V6 panel, and generated ~ 2.35 Gb of 1D sequence data from a MinION run, representing ~40x average coverage. We analysed the data using the resequencing analysis workflow (Fig. 2). Following basecalling, reads are mapped to the human exome reference sequence. Individual gene information is displayed, including coverage and read-accuracy distribution at that position. Future releases of this application will support the uploading of target regions, highlight known SNPs in the target regions and allow those SNPs to be displayed with a confidence value.

Fig. 3 Plots showing reads enriched for the BRCA1 gene and SNP-calling

Enrichment of Comprehensive Cancer panel genes, allowing detection of SNPs

We evaluated Agilent’s ClearSeq Comprehensive Cancer panel using DNA from two BRCA1 SNP-carriers with a positive family history of breast cancer (NA13708 and NA13710, Coriell NIGMS Human Genetic Cell Repository). We performed sequence capture as described, sequenced the library and basecalled reads using the 1D workflow. We aligned all reads to the reference sequence from Agilent, using BWA with standard parameters, and visualised reads with Savant (Fig. 3a). Figs. 3b and 3c show alignments of reads from exon 16 and exon 13, respectively, of the BRCA1 gene. Sequencing errors are distributed randomly, allowing SNPs to be seen clearly in the data. 

Fig. 4 Long reads mapping to exons 1–27 of NBPF1 after whole exome sequence capture

Long fragment capture protocol generates reads which can span multiple exons

We performed sequence capture on human genomic DNA, using Agilent’s SureSelect Human All Exon V6, using a randomly fragmented library which had been size-selected using the Blue Pippin automated gel fractionation system, leaving only fragments above 6 kb. The library was then amplified by PCR prior to performing hybrid capture. The library was sequenced on a single  run and the reads were mapped to the human genome (H19). Fig. 4 shows reads mapping to the neuroblastoma BPF1 gene, and it can be seen that multiple exons are spanned by many of the reads, meaning that both exon- and intron-specific variations can be detected and phased haplotypes can be obtained.

BWA and LAST have been tuned to work with nanopore reads

Burrow-Wheeler Aligner (BWA) for pairwise alignment between DNA sequences. BWA is a software package for mapping DNA sequences against a large reference genome, such as the human genome. It consists of three algorithms: BWA-backtrack, BWA-SW and BWA-MEM.

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NanoOk – Flexible, multi-reference software for pre- and post-alignment analysis of nanopore sequencing data, quality and error profiles

The recent launch of the Oxford Nanopore Technologies MinION Access Program (MAP) resulted in the rapid development of a number of open source tools aimed at extracting reads and yield information from the HDF5 format files produced by the platform.

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Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration.

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Sequencing ultra-long DNA molecules with the Oxford Nanopore MinION

Oxford Nanopore Technologies’ nanopore sequencing device, the MinION, holds the promise of sequencing ultra-long DNA fragments >100kb. An obstacle to realizing this promise is delivering ultra-long DNA molecules to the nanopores.

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A single chromosome assembly of Bacteroides fragilis strain BE1 from Illumina and MinION nanopore sequencing data

Second and third generation sequencing technologies have revolutionised bacterial genomics. Short-read Illumina reads result in cheap but fragmented assemblies, whereas longer reads are more expensive but result in more complete genomes.

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What’s in my pot? Real-time species identification on the MinION

Whole genome sequencing on next-generation instruments provides an unbiased way to identify the organisms present in complex metagenomic samples. However, the time-to-result can be protracted because of fixed-time sequencing runs and cumbersome bioinformatics workflows.

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nanopolish – nanopore sequence analysis and genome assembly software

Jared Simpson, University of Toronto

Release Date: 04-Sept-2015

A nanopore consensus algorithm using a signal-level hidden Markov model.

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Real-time selective sequencing on the MinION

The MinION replaces the conventional model of "sequence followed by analysis to final result" with instant access to data before the completion of a sequencing run. This instant access extends to the analysis of sequence "squiggle" data even before a read has finished traversing the nanopore.

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Progress at UC Santa Cruz: Long DNA fragments, tRNA and Modified Bases

Nanopore strand sequencing is uniquely suited to analysis of long DNA fragments and base modifications. In this presentation, we will discuss recent experiments that demonstrate 99% consensus accuracy for 150kb+ DNA fragments in single MinION runs.

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MinION sequencing of malaria parasites

The MinION device by Oxford Nanopore is the first portable sequencing device. MinION is able to produce very long reads (reads over 100~kBp were reported), however it suffers from high sequencing error rate.

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Nanopore sequencing for metagenomic diagnosis of infectious diseases

Unbiased diagnosis of all pathogens in a single test by metagenomic next-generation sequencing is now feasible, but has been limited to date by concerns regarding sensitivity and sample-to-answer turnaround times.

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Real-time nanopore sequencing and analysis of plant genomes in a tent in Snowdonia National Park

Joe Parker from Royal Botanic Gardens, Kew gives a talk at London Calling 2016 on how he used the MinION to analyse plant genomes in a tent in the middle of Snowdonia National Park.

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Genome sequencing a comprehensive communicable diseases solution in the making

Derrick Crook from University of Oxford gives a talk at London Calling 2016.

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A novel approach to elucidate the genomic structure of plants and pathogens

Alexander Wittenberg from KeyGene gives a talk at London Calling 2016.

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The Mystery of the Pink Lake

London Calling talk: The eXtreme Microbiome Project (XMP) Presents: The Mystery of the Pink Lake | Ken McGrath, Australian Genome Research Facility.

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Determining Exon Connectivity by Nanopore Sequencing

Short-read high-throughput DNA sequencing, though powerful, is limited in its ability to directly measure exon connectivity in mRNAs that contain multiple alternative exons located farther apart than the maximum read length.

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Fast and Sensitive Mapping of Nanopore Sequencing Reads with GraphMap

A presentation to the MinION Community by Professor Niranjan Nagarajan, Associate Director and Group Leader, Genome Institute of Singapore, and Adjunct Associate Professor in the Department of Computer Science, National University of Singapore.

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Oxford Nanopore MinION Applications: Kits and Tools, Genetics and Metagenomics

The Applications team at Oxford Nanopore has two overarching responsibilities: creation and development of sample and library preparation protocols for a wide variety of sample types, and undertaking biological projects which highlight the various strengths of Oxford Nanopore’s technology.

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Applications of Long-read Sequencing in Infectious Disease Genomics

Next generation sequencing technology has revolutionised the study of microbial genomics, but most large-scale studies have focused on short-read sequencing. This use of short-read sequencing has limitations however.

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Alignment, error correction and assembly of a eukaryote genome

Here we present our experiences working with MinION device. We discuss the performance, tools and algorithms available for downstream analysis; including alignment methods and parameters. Our interest in the MinION device is its ability to generate long reads for complex genome assembly.

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Sequence capture on the MinION

A presentation at London Calling 2015 by Daniel Fordham.

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Characterization, correction and de novo assembly of an Oxford Nanopore genomic dataset from Agrobacterium tumefaciens

The MinION is a portable single-molecule DNA sequencing instrument that was released by Oxford Nanopore Technologies in 2014, producing long sequencing reads by measuring changes in ionic flow when single-stranded DNA molecules translocate through the pores.

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De Novo Assembly of Human Herpes Virus Type 1 (HHV-1) Genome, Mining of Non-Canonical Structures and Detection of Novel Drug-Resistance Mutations Using Short- and Long-Read Next Generation Sequencing Technologies

Human herpesvirus type 1 (HHV-1) has a large double-stranded DNA genome of approximately 152 kbp that is structurally complex and GC-rich. This makes the assembly of HHV-1 whole genomes from short-read sequencing data technically challenging.

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Scaffolding and Completing Genome Assemblies in Real-time with Nanopore Sequencing

Genome assemblies obtained from short read sequencing technologies are often fragmented into many contigs because of the abundance of repetitive sequences.

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Clonal expansion of Escherichia coli ST38 carrying chromosomally-integrated OXA-48 carbapenemase gene

Many isolates of Escherichia coli carrying blaOXA-48 referred to Public Health England’s national reference laboratory during 2014 and 2015 shared similar pulsed-field gel electrophoresis (PFGE) profiles, despite coming from patients in multiple hospitals and regions.

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HPG pore: an efficient and scalable framework for nanopore sequencing data

The use of nanopore technologies is expected to spread in the future because they are portable and can sequence long fragments of DNA molecules without prior amplification.

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INC-Seq: Accurate single molecule reads using nanopore sequencing

Nanopore sequencing provides a rapid, cheap and portable real-time sequencing platform with the potential to revolutionise genomics. Several applications, including RNA-seq, haplotype sequencing and 16S sequencing, are however limited by its relatively high single read error rate (>10%).

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Nanopore sequencing detects structural variants in cancer

Despite advances in sequencing, structural variants (SVs) remain difficult to reliably detect due to the short read length (<300bp) of 2nd generation sequencing.

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Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool

Rapid sequencing of RNA/DNA from pathogen samples obtained during disease outbreaks provides critical scientific and public health information. However, challenges exist for exporting samples to laboratories or establishing conventional sequencers in remote outbreak regions.

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Streaming algorithms for identification of pathogens and antibiotic resistance potential from real-time MinION sequencing

The recently introduced Oxford Nanopore MinION platform generates DNA sequence data in real-time. This opens immense potential to shorten the sample-to-results time and is likely to lead to enormous benefits in rapid diagnosis of bacterial infection and identification of drug resistance.

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Evaluation of hybrid and non-hybrid methods for de novo assembly of nanopore reads

Recent emergence of nanopore sequencing technology set a challenge for the established assembly methods not optimised for the combination of read lengths and high error rates of nanopore reads. In this work we assessed how existing de novo assembly methods perform on these reads.

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MinION Analysis and Reference Consortium: Phase 1 data release and analysis

The advent of a miniaturised DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools.

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Oxford Nanopore sequencing, hybrid error correction, and de novo assembly of a eukaryotic genome

Monitoring the progress of DNA molecules through a membrane pore has been postulated as a method for sequencing DNA for several decades.

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MinION™ allied with 3D printed iChip – a workflow designed to let anyone discover new bacterial species in days

In this study, we adapt a protocol for the growth of previously uncultured environmental bacterial isolates, to make it compatible with whole genome sequencing.

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NanoOK: Multi-reference alignment analysis of nanopore sequencing data, quality and error profiles

Motivation: The Oxford Nanopore MinION sequencer, currently in pre-release testing through the MinION Access Programme (MAP), promises long reads in real-time from a cheap, compact, USB device.

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LINKS: Scalable, alignment-free scaffolding of draft genomes with long reads

Owing to the complexity of the assembly problem, we do not yet have complete genome sequences.

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Scaffolding of a bacterial genome using MinION nanopore sequencing

Second generation sequencing has revolutionized genomic studies. However, most genomes contain repeated DNA elements that are longer than the read lengths achievable with typical sequencers, so the genomic order of several generated contigs cannot be easily resolved.

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Nanopore sequencing for detection of pharmacogenomic variants and haplotypes

Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information.

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An epidemiological river metagenome based on MinION

River waters worldwide are impacted by disease-causing agents including bacteria, protists, flatworms, viruses, and harmful algae that derive from domestic sewage and farm runoff, and/or are emergent due to nutrient pollution and climate change.

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Genome assembly using Nanopore-guided long and error-free DNA reads

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.

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Improved data analysis for the MinION nanopore sequencer

Speed, single-base sensitivity and long read lengths make nanopores a promising technology for high-throughput sequencing.

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Long read nanopore sequencing for detection of HLA and CYP2D6 variants and haplotypes

Haplotypes are often critical for the interpretation of genetic laboratory observations into medically actionable findings. Current massively parallel DNA sequencing technologies produce short sequence reads that are often unable to resolve haplotype information.

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MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island

Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes.

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Think Small: Nanopores for Sensing and Synthesis

It is now possible to manipulate individual molecules using a nanopore to read DNA and proteins, or write DNA by inserting mini-genes into cells.

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Poretools: a toolkit for analyzing nanopore sequence data

Motivation: Nanopore sequencing may be the next disruptive technology in genomics, due to its ability to detect single DNA molecules without prior amplification, lack of reliance on expensive optical components, and the ability to sequence very long fragments.

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