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Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer*

Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak.

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Antimicrobial resistant Klebsiella pneumoniae carriage and infection in specialized geriatric care wards linked to acquisition in the referring hospital

Background. Klebsiella pneumoniae is a leading cause of extended-spectrum beta-lactamase (ESBL) producing hospital-associated infections, for which elderly patients are at increased risk. Methods.

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Towards a genomics-informed, real-time, global pathogen surveillance system

The recent Ebola and Zika epidemics demonstrate the need for the continuous surveillance, rapid diagnosis and real-time tracking of emerging infectious diseases.

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Detection of subclonal L1 transductions in colorectal cancer by long-distance inverse-PCR and Nanopore sequencing

Long interspersed nuclear elements-1 (L1s) are a large family of retrotransposons. Retrotransposons are repetitive sequences that are capable of autonomous mobility via a copy-and-paste mechanism.

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Campylobacter fetus meningitis confirmed by a 16S rRNA gene analysis using the MinION nanopore sequencer, South Korea, 2016

C. fetus should be considered a possible cause of bacterial meningitis, especially in immunocompromised patients with accompanying gastrointestinal symptoms. Nanopore sequencing of the 16S rRNA gene allowed the identification of C. fetus at the subspecies level.

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Nanopore Sequencing Reveals High-Resolution Structural Variation in the Cancer Genome

Acquired genomic structural variants (SVs) are major hallmarks of the cancer genome. Their complexity has been challenging to reconstruct from short-read sequencing data.

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On site DNA barcoding by nanopore sequencing

Note: the chemistry in this paper has since been superseded.

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Mapping DNA methylation with high-throughput nanopore sequencing

DNA chemical modifications regulate genomic function. We present a framework for mapping cytosine and adenosine methylation with the Oxford Nanopore Technologies MinION using this nanopore sequencer's ionic current signal. We map three cytosine variants and two adenine variants.
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Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates

Recent advances in nanopore sequencing technology have led to a substantial increase in throughput and sequence quality. Together, these improvements may permit real-time benchtop genomic sequencing and antimicrobial resistance gene detection in clinical isolates.
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De novo genome assembly and annotation of Australia's largest freshwater fish, the Murray cod (Maccullochella peelii), from Illumina and Nanopore sequencing read

One of the most iconic Australian fish is the Murray cod, Maccullochella peelii (Mitchell 1838), a freshwater species that can grow to ∼1.8 metres in length and live to age ≥48 years.
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Low bias RNA-seq: PCR-cDNA, PCR-free direct cDNA and direct RNA sequencing

PCR-based and PCR-free sequencing of full-length cDNA and RNA molecules gives lower bias and requires less data to cover transcripts than short-read sequencing

Fig. 1 RNA-seq workflows a) PCR-based cDNA, b) PCR-free direct cDNA, c) PCR-free direct RNA d) ERCC spike-in correlations for the three methods

Library preparation for long-read RNA-seq: full-length PCR- and PCR-free cDNA libraries using reverse transcription and strand-switching, and direct RNA sequencing

We have released three methods for the preparation of RNA-seq libraries. PCR-cDNA libraries are created by reverse transcription, strand-swtching and second-strand synthesis, followed by PCR and ligation of sequencing adapters (Fig. 1a). However, it is not necessary to amplify cDNA libraries prior to sequencing. In the direct cDNA protocol, sequencing adapters are attached directly to the doublestranded cDNA product, making library preparation considerably faster (Fig. 1b). Again, strand-switching is used to increase the proportion of full-length cDNAs. In addition to DNA, nanopores are also capable of sequencing RNA, without the need to reverse transcribe. In direct RNA sequencing, adapters are ligated onto the 3’ end of polyA-tailed RNA strands before sequencing (Fig. 1c). We evaluated all three RNA-seq approaches by making libraries from the ERCC spike-in panel (Fig. 1d, top panel is PCR-cDNA, middle panel is direct cDNA and bottom panel is direct RNA). This is a set of 92 polyadenylated RNAs, ranging from 250 to 2,000 nucleotides in length, which are present in the mixture at defined concentrations. Strong correlations were obtained between observed and expected read counts in all cases, with no evidence of length bias, showing that each method of RNA-seq is low bias. Interestingly, the PCR-cDNA protocol gave the strongest correlation (Spearman r = 0.98, p = 2.6e-61), in spite of the protocol including 15 cycles of PCR amplification. Of the three protocols, the PCR-cDNA protocol currently performs with the highest throughput.

Fig. 2 Quantitative assessment of bias a) GC bias b) length bias

Comparison of the influence of % GC and length across platforms and library preps

Sequencing workflows which incorporate amplification are vulnerable to sequence-specific biases. In particular, polymerases often struggle in regions with high or low GC contents. Additionally, amplification tends to favour shorter fragments. We investigated both types of bias by preparing yeast transcriptome libraries with all three of our protocols, and calculating correlations between GC content and read count (Fig. 3a) and between transcript length and read count (Fig. 3b). For comparison, we obtained a 100x PE Illumina dataset of the same sample, using a sequencing service. In all cases the bias in our data was lower than in the Illumina dataset, and most notably, GC bias was virtually absent.

Fig. 3 Data required to cover transcripts along 95% of their length, in terms of bases and reads

Less data is required to cover the same number of transcripts with long reads

To investigate the influence of long reads on the level of coverage required to cover the yeast transcriptome, using the same yeast datasets as those in Fig. 3, we mapped reads to the reference transcriptome. We took all transcripts which were covered along 95% of their length or more to be fully covered, and we counted the number of reads that were included by this definition for different numbers of bases and reads. Given that our technology allows us to sequence cDNA and RNA without fragmentation, the number of nanopore reads required to give the same level of coverage would be expected to be lower than for a short-read technology, but interestingly, the number of bases of sequence data required is also lower (Fig. 4).

First two cases of severe multifocal infections caused by Klebsiella pneumoniae in Switzerland: characterization of an atypical non-K1/K2-serotype strain causing liver abscess and endocarditis

Background

We describe the first two multifocal invasive infections due to Klebsiella pneumoniae recently observed in Switzerland.

Methods

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Heterogeneous Genetic Location of mcr-1 in Colistin-Resistant Escherichia coli Isolated from Humans and Retail Chicken Meat in Switzerland: Emergence of mcr-1-Carrying IncK2 Plasmids

We characterized the genetic environment of mcr-1 in colistin-resistant Escherichia coli strains isolated in Switzerland during 2014-2016 from humans (n=3) and chicken meat (n=6).
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Nanopore Long-Read Guided Complete Genome Assembly of Hydrogenophaga intermedia,

... and Genomic Insights into 4-Aminobenzenesulfonate, p-Aminobenzoic Acid and Hydrogen Metabolism in the Genus Hydrogenophaga.

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First Draft Genome Sequence of the Pathogenic Fungus Lomentospora prolificans (formerly Scedosporium prolificans)

Here we describe the sequencing and assembly of the pathogenic fungus Lomentospora prolificans using a combination of short, highly accurate Illumina reads and additional coverage in very long Oxford Nanopore reads.

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Profiling bacterial communities by MinION sequencing of ribosomal operons

Note: the chemistry used in this paper has since been superseded.

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de novo long-read assembly of a complex animal genome

Eukaryotic genome assembly remains a challenge in part because of the prevalence of complex DNA repeats. This is a particularly acute problem for holocentric nematodes because of the large number of satellite DNA sequences found throughout their genomes.

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A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION™ nanopore sequencer

Background

A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination.

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Real-time DNA barcoding in a remote rainforest using nanopore sequencing

Advancements in portable scientific instruments provide promising avenues to expedite field work in order to understand the diverse array of organisms that inhabit our planet.

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Metagenomic arbovirus detection using MinION nanopore sequencing

With its small size and low cost, the hand-held MinION sequencer is a powerful tool for in-field surveillance. Using a metagenomic approach, it allows non-targeted detection of viruses in a sample within a few hours.

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The Glycolytic Versatility of Bacteroides uniformis CECT 7771 and Its Genome Response to Oligo and Polysaccharides

Bacteroides spp. are dominant components of the phylum Bacteroidetes in the gut microbiota and prosper in glycan enriched environments.

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Rapid MinION metagenomic profiling of the preterm infant gut microbiota to aid in pathogen diagnostics

The Oxford Nanopore MinION sequencing platform offers direct analysis of DNA reads as they are generated, which combined with its low cost, low power and extremely compact size, makes the device attractive for in-field or clinical deployment, e.g. rapid diagnostics.

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npInv: accurate detection and genotyping of inversions mediated by non-allelic homologous recombination using long read sub-alignment

Detection of genomic inversions remains challenging. Many existing methods primarily target inversions with a non repetitive breakpoint, leaving inverted repeat (IR) mediated non-allelic homologous recombination (NAHR) inversions largely unexplored.

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Accurate typing of class I human leukocyte antigen by Oxford nanopore sequencing

Oxford Nanopore Technologies' MinION has expanded the current DNA sequencing toolkit by delivering long read lengths and extreme portability.

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Nanopore sequencing enables near-complete de novo assembly of Saccharomyces cerevisiae reference strain CEN.PK113-7D

The haploid Saccharomyces cerevisiae strain CEN.PK113-7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C.

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The potential impact of nanopore sequencing on human genetics.

Nanopore sequencing has been available to customers for a little over three years. Recently the milestone of sequencing and assembling a human genome on this platform was achieved for the first time.

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Linear Assembly of a Human Y Centromere using Nanopore Long Reads

The human genome reference sequence remains incomplete due to the challenge of assembling long tracts of near-identical tandem repeats, or satellite DNAs, that are highly enriched in centromeric regions.

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Nanopore sequencing of full-length BRCA1 mRNA transcripts reveals co-occurrence of known exon skipping events

Laboratory assays evaluating the effect of DNA sequence variants on BRCA1 mRNA splicing may contribute to classification by providing molecular evidence.

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Multiplexed nanopore sequencing of HLA-B locus in Māori and Polynesian samples

The human leukocyte antigen (HLA) system is a gene family that encodes the human major histocompatibility complex (MHC). HLA-B is the most polymorphic gene in the MHC class I region, comprised of 4,765 HLA-B alleles (IPD-IMGT/HLA Database Release 3.28).

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Comparative analysis of targeted long read sequencing approaches for characterization of a plant’s immune receptor repertoire

Background

The Oxford Nanopore Technologies MinION™ sequencer is a small, portable, low cost device that is accessible to labs of all sizes and attractive for in-the-field sequencing experiments.

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Using MinION to characterize dog skin microbiota through full-length 16S rRNA gene sequencing approach

The most common strategy to assess microbiota is sequencing specific hypervariable regions of 16S rRNA gene using 2nd generation platforms (such as MiSeq or Ion Torrent PGM).

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Detection of active transposable elements in Arabidopsis thaliana using Oxford Nanopore Sequencing technology

Note: this publication uses R7 chemistry, which has since been superseded with R9 series.

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Efficient data structures for mobile de novo genome assembly by third-generation sequencing

Mobile/portable (third-generation) sequencing technologies, including Oxford Nanopore’s MinION and SmidgION, are revolutionizing once again –after the advent of high-throughput sequencing– biomedical sciences.

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Hand-held rapid whole genome nanopore sequencing to predict neisseria gonorrhoeae antibiotic susceptibility: steps towards clinic based tailored antimicrobial therapy

Next generation sequencing can accurately predict antibiotic susceptibility in Neisseria gonorrhoeae (NG) allowing preservation of first-line treatments in the face of widespread antimicrobial resistance (AMR).

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Sharing of carbapenemase-encoding plasmids between Enterobacteriaceae in UK sewage uncovered by MinION sequencing

Dissemination of carbapenem resistance among pathogenic Gram-negative bacteria is a looming medical emergency. Efficient spread of resistance within and between bacterial species is facilitated by mobile genetic elements.

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Sequencing and phasing cancer mutations in lung cancers using a long-read portable sequencer

Here, we employed cDNA amplicon sequencing using a long-read portable sequencer, MinION, to characterize various types of mutations in cancer-related genes, namely, EGFR, KRAS, NRAS and NF1. For homozygous SNVs, the precision and recall rates were 87.5% and 91.3%, respectively.

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Mutational analysis in BCR-ABL1 positive leukemia by deep sequencing based on nanopore MinION technology

We report a third-generation sequencing assay on nanopore technology (MinION) for detecting BCR-ABL1 KD mutations and compare the results to a Sanger sequencing(SS)-based test in 24 Philadelphia-positive (Ph +) leukemia cases.

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De novo yeast genome assemblies from MinION, PacBio and MiSeq platforms

Long-read sequencing technologies such as Pacific Biosciences and Oxford Nanopore MinION are capable of producing long sequencing reads with average fragment lengths of over 10,000 base-pairs and maximum lengths reaching 100,000 base- pairs.

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Nanopore-based single molecule sequencing of the D4Z4 array responsible for facioscapulohumeral muscular dystrophy

Subtelomeric macrosatellite repeats are difficult to sequence using conventional sequencing methods owing to the high similarity among repeat units and high GC content. Sequencing these repetitive regions is challenging, even with recent improvements in sequencing technologies.

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Same-day genomic and epigenomic diagnosis of brain tumors using real-time nanopore sequencing

Molecular classification of cancer has entered clinical routine to inform diagnosis, prognosis, and treatment decisions. At the same time, new tumor entities have been identified that cannot be defined histologically.

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Serotyping dengue virus with isothermal amplification and a portable sequencer

The recent development of a nanopore-type portable DNA sequencer has changed the way we think about DNA sequencing. We can perform sequencing directly in the field, where we collect the samples.

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Establishment and cryptic transmission of Zika virus in Brazil and the Americas

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil

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High-quality de novo genome assembly of the Dekkera bruxellensis UMY321 yeast isolate using Nanopore MinION sequencing

Genetic variation in natural populations represents the raw material for phenotypic diversity. Species-wide characterization of genetic variants is crucial to have a deeper insight into the genotype-phenotype relationship.

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Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

Background: The miniaturised and portable DNA sequencer MinIONTM has demonstrated great potential in different analyses such as genome-wide sequencing, pathogen outbreak detection and surveillance, human genome variability, and microbial diversity

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Measuring Soil Health With Nanopore Sequencing

If a farmer wants to understand the health and fertility of their soil, they could send a sample to a lab for chemical analysis. This produces useful data, including levels of: 1) nitrogen, 2) phosphorus, 3) potassium and 4) pH.

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Resolving plasmid structures in Enterobacteriaceae using the MinION nanopore sequencer: assessment of MinION and MinION/Illumina hybrid data assembly approaches

This study aimed to assess the feasibility of using the Oxford Nanopore Technologies (ONT) MinION long-read sequencer in reconstructing fully closed plasmid sequences from eight Enterobacteriaceae isolates of six different species with plasmid populations of varying complexity.

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High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell

While many evolutionary questions can be answered by short read re-sequencing, presence/absence polymorphisms of genes and/or transposons have been largely ignored in large-scale intraspecific evolutionary studies.

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Deleterious ABCA7 mutations and transcript rescue mechanisms in early onset Alzheimer’s disease

Premature termination codon (PTC) mutations in the ATP-Binding Cassette, Sub-Family A, Member 7 gene (ABCA7) have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer’s disease (LOAD).

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De novo whole-genome assembly of a wild type yeast isolate using nanopore sequencing

Background: The introduction of the MinIONTM sequencing device by Oxford Nanopore Technologies may greatly accelerate whole genome sequencing.

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Deep Sequencing: Intra-Terrestrial Metagenomics Illustrates The Potential Of Off-Grid Nanopore DNA Sequencing

Genetic and genomic analysis of nucleic acids from environmental samples has helped transform our perception of the subsurface as a major reservoir of microbial novelty. Many of the microbial taxa living in the subsurface are under-represented in culture-dependent investigations.

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Strand-specific preparation of full-length PCR-based and PCR-free cDNA libraries by strand-switching

Strand-specific methods for PCR-based and PCR-free synthesis of full-length cDNA molecules, giving quantitative results, and semi-specific RT-PCR for targeting fusion transcripts 

Fig. 1 PCR-based strand-switching a) laboratory workflow b) ERCC control-mix counts

Strand-switching protocol for preparation of full-length 1D cDNA libraries

We have introduced a PCR-based strand-switching protocol for 1D cDNA sequencing (Fig. 1a). Following first-strand synthesis, reverse transcriptases add 1–3 non-templated Cs to the 3’ end of the cDNA strand. By including an additional primer in the RT reaction which anneals to the non-templated Cs, we can make the reverse transcriptase switch templates, to extend opposite this primer. This incorporates a PCR-priming sequence to the end of full-length cDNAs. After PCR, sequencing adapters are attached. We validated this protocol using the ERCC control mix and obtained excellent correlation with the expected values, regardless of the length of the RNA strands (Spearman’s rho = 0.98; p = 2.6e-61; Fig. 1b), showing quantitative performance.

Fig. 2 PCR-free ‘direct cDNA’ a) laboratory workflow b) ERCC control-mix counts

Direct cDNA protocol for preparation of full-length 1D PCR-free cDNA libraries

It is not necessary to amplify libraries in any way in order to sequence them using our technology. With this in mind, we have developed a PCR-free ‘direct’ cDNA protocol. Here, a first strand cDNA molecule is synthesised, the original RNA is digested, a second strand cDNA is synthesised and sequencing adapters are attached (Fig. 2a). As with the PCR-based protocol, we validated the direct protocol by preparing a library from the ERCC control mix and counting the sequences obtained from each RNA in the mixture. As might be expected from an amplification-free workflow, we obtained a very good correlation with the expected values, regardless of the length of the RNA strands (Spearman’s rho = 0.96; p = 1.4e-52; Fig. 2b).

Fig. 3 Yeast transcriptome a) distance and coverage b) reference coverage c) strand-specificity

Strand-switching showing strand-specificity and enrichment of full-length cDNAs

We applied both PCR and PCR-free protocols to the Saccharomyces cerevisiae S228C transcriptome. Both protocols give good coverage of entire transcripts in single reads (Fig. 2a), and the overwhelming majority of alignments have a coverage value close to 1, indicating that alignments tend to cover full transcripts (Fig. 2b). In both protocols, cDNA extension primes from the eukaryotic poly-A tail, and the signature is retained in the sequences, allowing us to work out the strand in the gDNA from which the transcript was synthesised. This gives us the ability to identify sense and antisense transcripts unambiguously. Fig. 2c shows examples of directionality of full-length reads for a family of transcripts in the SIRV standard panel.
 

Fig. 4 Targeted detection a) chr22 translocations b) detection protocol c) fusion breakpoint

Sequencing semi-specific RT-PCR products enables characterisation of fusion genes

The q12 region of human chromosome 22 can be involved in several different translocation events (Fig 4a). In each of these, a fusion gene is formed by addition of chromosomal material from one of the fusion partners onto exon 7/8 of the EWSR1 gene on derived chromosome 22. These fusion genes lead to different types of cancer. We reverse-transcribed total RNA from a patient with a known translocation affecting the EWSR1 gene, using a poly TVN primer, and we amplified semi-specifically. The wild-type EWSR1 amplicon and fusion amplicons were 2.2 and 3.1 kb respectively. Sequencing of the amplicons, and alignment to the EWSR1 reference allowed us to pinpoint the position of the breakpoint and to identify the fusion partner. 

Using long nanopore reads to delineate structural variants (SVs) in the human genome

SVs, including large deletions, duplications, inversions, translocations and copy number changes are abundant in large genomes, and require long reads for precise characterisation.

Fig. 1 Structural variation a) classes b) variant size and frequency in the human genome

Structural variation: large inversions, deletions, duplications and translocations

Structural variation (SV) refers to inversions, insertions, deletions and translocations > 1 kb in length (Fig. 1a). SV encompasses millions of bases of DNA per human genome, can span tens of kilobases, containing entire genes and their regulatory regions (Fig. 1b), and contributes substantially to genome variation. SV can alter the copy number of dosage-sensitive genes, can unmask recessive alleles and can disrupt the integrity or regulation of a gene, all of which can cause genetic disease. The study of SVs is challenging because they frequently arise in repetitive regions of the genome, and can have highly complex structures. Short-read sequencing technologies cannot span long SVs, leading to incomplete reference assemblies. 

Fig. 2 Read length a) typical distribution b) assembly c) mapped long human MinION reads

Nanopore sequencing can give extremely long reads without size selection

The read length that can be obtained from nanopore sequencing is limited only by the integrity of the DNA extracted from the sample and the care taken during library preparation. The read- length distribution corresponds closely to the fragment length distribution of the sample DNA. When starting with high-molecular weight genomic DNA, it is straightforward to obtain reads that are tens of kilobases in length (Fig. 2a). The longer the sequence read, the longer the repetitive region or SV that can be resolved, allowing the correct structure of the variant to be elucidated (Fig. 2b). Recent increases in throughput make it realistic to sequence whole human genomes on a MinION (Fig. 2c). 

Fig. 3 Confirmation of LEO1 breakpoints and parental origin with nanopore reads

Deletion of a regulatory element in autistic patients validated by long nanopore reads

To demonstrate the utility of long nanopore reads in resolving structural variants, we amplified and barcoded patient and wild-type alleles from three families with known deletions in the LEO1 locus on chromosome 15, and sequenced them on a flowcell. Deletion amplicons were approximately 10 kb in length, and the amplifiable wild-type amplicons spanned up to 20 kb. LEO1 encodes an RNA polymerase-associated protein which is expressed during foetal brain development. For the deletions, we created consensus reference haplotypes using Nanopolish and realigned reads to these references for SNP-calling with mummer. All three deletions, as well as the parental origin, were successfully validated by the nanopore reads (Fig. 3). 

Fig. 4 Detection of SVs by whole-genome sequencing a) mapped reads b) SV resolution

Using long-read whole-genome sequencing to resolve SVs in the human genome

One individual who participated in the autism spectrum disorder study described in Fig. 3 had been diagnosed with depression/anxiety. She appeared to have an SV in chromosome 10 which had been identified as a complex break-end by Lumpy analysis of paired-end Illumina data. The SV was not found in the individual’s parents, so was taken to be de novo, but the precise structure was unclear. We performed whole-genome library prep using an LSK108 kit, and sequenced the library on a FLO-MIN106 flowcell, generating approximately 24 Gb of sequence data. The long reads allowed us to fully resolve the variant, and nanopore data was phased using WhatsHap, revealing the individual’s mother to be the parent of origin of the SV. 

Multiplexed quantification of protein panels by nanopore sequencing of reporter oligonucleotides

Adapting a sandwich enzyme-linked immunosorbent assay (ELISA) to give a DNA-based readout enables an accurate PCR-free and multiplexed method of protein quantification

Fig. 1 Workflow diagram for nanopore protein quantification

PCR-free workflow for ELISA-based protein quantification nanopore assay

The sandwich ELISA is a sensitive, specific and robust method for the quantification of proteins present in a biological sample. A primary antibody is immobilised on a solid surface, and when an antigen is present, this binds to the primary antibody. A secondary antibody then binds to the antigen. In our adapted version of the protocol this secondary antibody has a biotin group attached to the free end of one heavy chain. Following antibody/antigen complex formation, streptavidin is added. Streptavidin is capable of binding four biotin groups, meaning that when a streptavidin molecule binds to a heavy chain, an average of three binding sites are still available. We add reporter oligonucleotides to these available binding sites (Fig. 1). 

Fig. 2 a) PSA b) Sandwich ELISA sequencing assay showing quantitative results

Quantitative uniplex modified sandwich ELISA protocol demonstrated on PSA

To demonstrate the effectiveness of this approach in quantifying a protein, we developed an assay for PSA, a protein which is frequently described as a biomarker for prostate cancer. Blood PSA concentrations greater than 4 ng/ml are considered to be ‘elevated’ and warrant further tests (Fig. 2a). We took a range of PSA concentrations in duplicate, both above and below 4 ng/ml, and performed the method outlined in Fig. 1, using reporter strands which were barcoded differently for each PSA concentration. All eluted strands were then pooled and sequenced together on a single flowcell, and read counts for each reporter oligo were reported. We obtained a good correlation between reporter read count and PSA concentration (Fig. 2b).

Fig. 3 Multiplexed protein quantification a) 3 protein standards b) lung cancer biomarker panel

Multiplexed quantification of proteins by nanopore sequencing

It is typically necessary to quantify panels of two or more ‘biomarker’ proteins in a single assay to measure disease progress. To see if this would be possible, we designed assays for several proteins. Antibody capture was performed separately for each protein over a range of concentrations, and antibody-protein conjugates were labelled with reporter oligonucleotides, using a different reporter for each protein, and a different barcode for each protein concentration. The eluted reporters were pooled and sequenced together on one flowcell. Fig. 3a shows titration curves for three protein standards and Fig. 3b shows results for a lung cancer biomarker panel. The sensitivity of each assay exceeds that required to measure these proteins at their physiological concentrations. 

Fig. 4 Combined testing of biomarker proteins, transcriptome and exome on single flowcell

Combined nanopore testing of exome, transcriptome and biomarkers

Although for the experiments presented here we only multiplexed at the level of sequencing, pre-assembling the secondary antibody/reporter oligonucleotides will enable several proteins to be assayed in a single reaction. In addition, this will allow us to balance the reporter read count for all the different proteins in a panel, even though they may occur at vastly different concentrations in serum. The relatively low number of reporter reads needed for sensitive protein quantification means that a large biomarker panel could be quantified on a single flowcell while that same flowcell is used to sequence that person’s exome and transcriptome simultaneously (Fig. 4a). This could be used to provide a comprehensive assessment of the status of a person’s health, and when combined with VolTRAX, could be performed outside of the laboratory (Fig. 4b). 

Combined pre-implantation genetic screening (PGS) for aneuploidy and haplotyping of the ANXA5 gene

Screening for chromosomal aneuploidy and miscarriage predisposition in IVF embryos in real time means no freeze/thawing and reduces the waiting time for embryo transplantation.

Fig. 1 Workflow for combined aneuploidy screening and single-locus testing

PGS and PGD improve IVF success rate by screening for abnormalities

PGS is the process of screening an IVF embryo for aneuploidy by counting chromosome number, using low-coverage sequencing of the whole genome. Conversely, preimplantation genetic diagnosis (PGD) tests a single gene, and requires higher coverage of that region, for variant-calling. PCR amplification of the single region for a limited number of cycles enriches for the target region, without overwhelming the whole-genome template. Following amplification, sequencing adapters can be attached to both the amplicon and the accompanying whole-genome template, creating a combined PGS-PGD sequencing library (Fig. 1). 

Fig. 2 ANXA5 a) locus and variants b) haplotyping results. Blue = wild-type, green = M2

ANXA5 M2 haplotype is associated with increased risk of miscarriage

The human ANXA5 gene, situated on chromosome 4, encodes a calcium-dependent phospholipid binding protein, which acts as a placental anticoagulant. A variant haplotype of ANXA5 contains 4 nucleotide substitutions which lie within the space of 57 nucleotides in the promoter (Fig. 2a). These substitutions reduce the activity of the promoter substantially, and if the embryo inherits the M2 haplotype from either parent, the risk of miscarriage increases substantially. Rather than testing the parents, by amplifying across the region in blastocyst DNA, followed by sequencing, we are able to identify embryonic ANXA5 haplotypes (Fig. 2b).

Fig. 3 Results of combined PGS and ANXA5 haplotyping in two aneuploid embryos

Combined aneuploidy screening and ANXA5 haplotyping of blastocyst DNA

We extracted genomic DNA from 1–3 cells taken from thirty 5-day-old IVF blastocysts and performed whole genome amplification (WGA) of the DNA. Combined PGS/ANXA5 libraries were created and the barcoded products were quantified before being pooled and sequenced, 6 samples per flowcell. Sequence reads were analysed using our PGS bioinformatics pipeline and results of both aneuploidy screening and ANXA5 haplotyping for two samples are shown in Fig. 3. Our ploidy calls for each sample were in full concordance with the CGH results, and the ANXA5 haplotype calls of each sample were verified by capillary sequencing.

Fig. 4 Higher-resolution PGS screening a) read depth and resolution b) Cri du chat syndrome

Aneuploidy and higher-resolution analyses from low-coverage nanopore data

We took a higher-coverage PGS dataset and downsampled the data, to find the minimal number of reads required to robustly identify the ploidy level of a sample. We calculated that 50,000 reads of 500 nt in length are required, equating to approximately 0.01x, or 30 Mb, per sample (Fig. 4a). Our calculations also indicate that without significantly greater coverage, we can detect smaller-scale abnormalities than whole-chromosome aneuploidies. To test this, we prepared the same library type from a cell line carrying the Cri du Chat deletion. The partial deletion of the short arm of chromosome 5 is clearly visible, along with several cell-line artefacts (Fig. 4b). 

Rapid DNA Re-Identification for Cell Line Authentication and Forensics

DNA re-identification is used for a broad range of applications, ranging from cell line authentication to crime scene sample identification. However, current re-identification schemes suffer from high latency.

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Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing

The ribosome small subunit is expressed in all living cells. It performs numerous essential functions during translation, including formation of the initiation complex and proofreading of base-pairs between mRNA codons and tRNA anticodons.

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Mapping and phasing of structural variation in patient genomes using nanopore sequencing

Despite improvements in genomics technology, the detection of structural variants (SVs) from short-read sequencing still poses challenges, particularly for complex variation.

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De novo Assembly of a New Solanum pennellii Accession Using Nanopore Sequencing

Recent updates in sequencing technology have made it possible to obtain Gigabases of sequence data from one single flowcell. Prior to this update, the nanopore sequencing technology was mainly used to analyze and assemble microbial samples.

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Nanopore sequencing and assembly of a human genome with ultra-long reads

Nanopore sequencing is a promising technique for genome sequencing due to its portability, ability to sequence long reads from single molecules, and to simultaneously assay DNA methylation.

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Nanopore detection of bacterial DNA base modifications

The common bacterial base modification N6-methyladenine (m6A) is involved in many pathways related to an organism's ability to survive and interact with its environment.

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Nanopore Long-Read RNAseq Reveals Widespread Transcriptional Variation Among the Surface Receptors of Individual B cells

Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single cell level.

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Fast and sensitive mapping of nanopore sequencing reads with GraphMap

Realizing the democratic promise of nanopore sequencing requires the development of new bioinformatics approaches to deal with its specific error characteristics.

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Real-Time DNA Sequencing in the Antarctic Dry Valleys Using the Oxford Nanopore Sequencer

The ability to sequence DNA outside of the laboratory setting has enabled novel research questions to be addressed in the field in diverse areas, ranging from environmental microbiology to viral epidemics.

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Nanopores allow direct sequencing of RNA strands, giving full-length reads with low bias

Complete RNA strands can be sequenced on the MinIONTM, GridIONTM and PromethIONTM using a simple library prep, without the need to convert to double-stranded DNA.

Fig. 1 Direct RNA sequencing a) library-prep workflow b) ‘squiggle’ c) alignment

Direct RNA offers native-strand sequencing, quick library prep and full-length transcripts

Nanopores are the only sequencing technology which can sequence an RNA strand directly, rather than analysing the products of reverse transcription and PCR reactions. In the workflow shown in Fig. 1a, an adapter is attached to the poly-A tail at the 3’ end of the RNA strand. This adapter is pre-loaded with a motor protein. This protein controls the speed of translocation of the RNA strand through the nanopore. Fig. 1b shows the raw data produced by translocation of a complete 1,500 nt transcript through the nanopore. The poly-A tail can be seen close to the start of the read since, unlike DNA, RNA is sequenced 3’ end first. Fig. 1c shows a section of a Direct RNA read obtained from a yeast transcriptome dataset, aligned to the reference.

Fig. 2 Direct RNA sequencing a) single-read accuracy, b) alignment coverage c) read length

Increases in throughput allow generation of transcriptome-wide, full-length datasets

We took poly-A-selected RNA from Saccharomyces cerevisiae S228C and prepared a whole transcriptome library using the protocol outlined in Fig. 1. The modal accuracy of these reads was > 90% (Fig. 2a). Mapping the reads back to the reference transcriptome and calculating the proportion of each transcript that is covered by each read indicates that we obtain a high proportion of full-length reads (Fig. 2b). This is in spite of the fact that the protocol does not currently contain a step to enrich for full-length transcripts. When the read-lengths produced by sequencing the yeast Direct RNA library are compared to those from a strand-switching cDNA dataset, the distributions overlap substantially (Fig. 2c). 

Fig. 3 Yeast transcriptome a) Circos plot b) correlation with Illumina data c) isozyme detection

RNA data correlates well with short reads and identifies isozymes unambiguously

We obtained a 100-base paired-end Illumina dataset of the same yeast sample, aligned the Direct RNA and Illumina reads to the Saccharomyces cerevisiae genome, and calculated the log-fold change in coverage between the Direct RNA and Illumina datasets. The number of Direct RNA reads mapped to the yeast genome using GMAP 22 was 2045748 (63.43%), while the number of Illumina RNA-seq reads mapped by GSNAP was 708592030 (98.22%, Figs. 3a and 3b). Direct RNA gene-coverage corresponds well with the Illumina results, with an overall correlation of 0.73. Two of the identified transcripts were isozymes of GAPDH, forms of the same gene but which reside at different positions in the genome. Even though the homology of the genes is higher than the current accuracy, we could map reads without ambiguity (Fig. 3c). 

Fig. 4 Reads of spike-ins showing a) splice variant detection b) quantitative measurement

Analysis of spike-ins demonstrates both the ability to detect splice variants and low bias

The long reads generated by Direct RNA sequencing on our nanopore devices should allow straightforward detection of splice variants. We investigated this using Lexogen’s SIRV panel, and were able to detect the majority of variants from the panel (Fig. 4a). Having no amplification means that Direct RNA sequencing should show low quantitative bias. However, many RNAs in the SIRV panel are highly similar, resulting in some mismapping. When the more dissimilar RNAs in the ERCC panel are used, the read counts match the expected values extremely closely (Fig. 4b, Spearman r = 0.97; p = 5.9e-56). We anticipate that improvements in the accuracy of Direct RNA sequencing will enhance our ability to map highly similar splice variants and will decrease bias further still.

Non-invasive diagnosis of cutaneous leishmaniasis by the direct boil loop-mediated isothermal amplification method and MinION™ nanopore sequencing

Cutaneous leishmaniasis (CL) is gaining attention as a public health problem. We present two cases of CL imported from Syria and Venezuela in Japan.

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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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Cas9-Assisted Targeting of CHromosome segments (CATCH) for targeted nanopore sequencing and optical genome mapping

Variations in the genetic code, from single point mutations to large structural or copy number alterations, influence susceptibility, onset, and progression of genetic diseases and tumor transformation.

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The use of Oxford Nanopore native barcoding for complete genome assembly

The Oxford Nanopore Technologies MinIONTM is a mobile DNA sequencer that can produce long read sequences with a short turn-around time.

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Picopore: A tool for reducing the size of Oxford Nanopore Technologies' datasets without losing information.

A tool for reducing the size of Oxford Nanopore Technologies' datasets without losing information.

Options:

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A portable system for metagenomic analyses using nanopore-based sequencer and laptop computers can realize rapid on-site determination of bacterial compositions

We developed a portable system for metagenomic analyses consisting of nanopore technology-based sequencer, MinION, and laptop computers, and assessed its potential ability to determine bacterial compositions rapidly.

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Annotated mitochondrial genome with Nanopore R9 signal for Nippostrongylus brasiliensis

Nippostrongylus brasiliensis, a nematode parasite of rodents, has a parasitic life cycle that is an extremely useful model for the study of human hookworm infection, particularly in regards to the induced immune response.

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Evaluation of Oxford Nanopore MinION Sequencing for 16S rRNA Microbiome Characterization

In this manuscript we evaluate the potential for microbiome characterization by sequencing of near-full length 16S rRNA gene region fragments using the Oxford Nanopore MinION (hereafter Nanopore) sequencing platform. We analyzed pure-culture E. coli and P.

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Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads.

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poRe GUIs for parallel and real-time processing of MinION sequence data

Motivation: Oxford Nanopore's MinION device has matured rapidly and is now capable of producing over one million reads and several gigabases of sequence data per run.

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Whole genome sequencing and assembly of a Caenorhabditis elegans genome with complex genomic rearrangements using the MinION sequencing device

Advances in 3rd generation sequencing have opened new possibilities for benchtop whole genome sequencing. The MinION is a portable device that uses nanopore technology and can sequence long DNA molecules.

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Identification of a novel species of papillomavirus in giraffe lesions using nanopore sequencing

Papillomaviridae form a large family of viruses that are known to infect a variety of vertebrates, including mammals, reptiles, birds and fish. Infections usually give rise to minor skin lesions but can in some cases lead to the development of malignant neoplasia.

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NA12878 Human Reference on Oxford Nanopore MinION

We have sequenced the CEPH1463 (NA12878/GM12878, Ceph/Utah pedigree) human genome reference standard on the Oxford Nanopore MinION using 1D ligation kits (450 bp/s) using R9.4 chemistry (FLO-MIN106).

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Sequencing human genomes with nanopore technology

Michael is co-head of science at Genomics plc and Professor of Genetics at King’s College London. His work focusses on the application of contemporary genomic technologies to detect genetic variation and evaluate its role in human disease.

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De novo Identification of DNA Modifications Enabled by Genome-Guided Nanopore Signal Processing

Advances in single molecule sequencing technology have enabled the investigation of the full catalogue of covalent DNA modifications.

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Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples.

Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks.

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Sequencing the gigabase plant genome of the wild tomato species Solanum pennellii using Oxford Nanopore single molecule sequencing

Recent updates in Oxford Nanopore technology (R9.4) have made it possible to obtain GBases of sequence data from a single flowcell.

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VolTRAX: a versatile, programmable, portable device for sample and library preparation

VolTRAX is designed to extract nucleic acids and prepare sequencing libraries from biological samples, enabling consistent library preparation even in non-laboratory environments

Fig. 1 The VolTRAX device

VolTRAX: automated sample and library preparation without human intervention

Oxford Nanopore is developing VolTRAX – a small device designed to perform all of the molecular biological manipulations required to convert a raw biological sample to a form ready for analysis on a nanopore sensing device, without the need for human intervention (Fig. 1). Ultimately, the device will be dockable with MinIONTM, GridIONTM or PromethIONTM, allowing fully automated sequencing in both laboratory and non-laboratory environments. The technology behind VolTRAX utilises an array of pixels. By applying a charge, reagent and sample droplets are moved in a path programmed by software, allowing the separate sample and library preparation processes to be performed sequentially.

Fig. 2 Library prep a) read-length distribution b) throughput c) multiplexing d) quantification

Library prep workflows show equivalent performance on VolTRAX and in tubes

We are adapting all of our library preparation protocols to work on VolTRAX. When a library is prepared on VolTRAX, we obtain a similar read-length distribution to that seen when the prep is performed in a tube (Fig. 2a). Additionally, the reactions have been optimised extensively on VolTRAX to give equivalent performance to the highest-throughput tube prep (Fig. 2b). Having several reagent ports on the VolTRAX cartridge allows us to perform multiple barcoded library preps in parallel. We obtain very similar read counts from each barcode, showing the reproducibility of library preparation on VolTRAX (Fig. 2c). We are also incorporating fluorescence measurement into the device, to allow quantification of DNA and RNA (Fig. 2d).

Fig. 3 Sample to result: using VolTRAX in species-identification workflows

Cell lysis, 16S PCR and library prep performed on VolTRAX, and CO1 species ID

We performed lysis of an E. coli culture, DNA purification using SPRI beads, 18 rounds of PCR on the extract to amplify the ~1.5 kb 16S region, and attachment of sequencing adapters (Fig. 3a). All steps were performed on VolTRAX. Finally, we removed the prepared library and sequenced it to confirm the identity of the species using our 16S species identification workflow (Fig. 3b). Some samples are more difficult to extract DNA from, and here it can be helpful to use bead-beating prior to DNA isolation. We extracted DNA from the invasive ladybird species Harmonia axyridis by bead-beating, and loaded the crude extract onto VolTRAX, where we amplified a 650 bp region of the CO1 gene and attached sequencing adapters (Fig. 3c). BLAST analysis of the reads confirmed the identity of the sample (Fig. 3d).

Fig. 4 Whole-genome amplification on VolTRAX a) workflow b) WIMP identification

Whole-genome amplification on VolTRAX enables low-input sequencing

In situations where small amounts of input DNA are available, whole-genome amplification (WGA) can be used to increase the mass of DNA. With this in mind, we adapted a WGA protocol for use on VolTRAX. In contrast to the standard protocol, a proteinase K digestion and SPRI clean-up are performed after the WGA reaction. The DNA is then fragmented and adapters are added using our rapid library prep protocol (Fig. 4a). We tested the protocol on a sample of soil taken from the Oxford Science Park, where the ONT labs are located. The soil was resuspended in water, filtered and the filtrate loaded onto VolTRAX for WGA, quantification and library preparation. As the library was being sequenced, we used the WIMP analysis tool to identify the species present, in real time. These included bacteria, fungi and viruses (Fig. 4b).

Barcode of life: simple laboratory and analysis workflows for 16s and CO1 analysis

Genus- and species-level identification by 16s or CO1 analysis made easy using a rapid laboratory protocol for adapter attachment and new data-analysis workflow

Fig. 1 Laboratory workflow for barcoded 16S and CO1 sequencing

Locus-specific PCR coupled with rapid adapter attachment for 16S and CO1

It is often desirable to be able to identify the species present in a complex mixture. This can be achieved by amplifying the bacterial 16S or mammalian cytochrome oxidase (CO1) loci, and comparing the results with a reference database. PCR amplification of specific loci can allow enrichment of the target region in the presence of a large background of other organisms. By modifying the 5’ ends of standard PCR primers used for amplification of these loci we have developed a protocol that attaches our sequencing adapters to the amplicons in approximately 5 minutes, enabling more rapid species identification (Fig. 1). 

Fig. 2 Analysis report for species identification, shown here for Staphylococcus 16S

Analysis workflow and report simplifies amplicon-based species identification

In the 16S analysis workflow, reads are compared to the NCBI 16S bacterial database using the Basic Local Alignment Search Tool (BLAST), immediately after each read has been basecalled. To validate the workflow, we prepared 1D 16S libraries by PCR amplification of the ZymoBIOMICS Microbial Community DNA Standard, sequenced the libraries on MinIONTM flowcells and passed the basecalled results through the analysis workflow (Fig. 2). We were able to classify all eight bacteria in the mock community to genus level. We calculated the precision at genus level to be 99% for 1D data. 

Fig. 3 Comparison of whole-genome and 16S identification at a) genus and b) species levels

16S compared to whole genome identification at genus and species levels

We generated whole genome and 16S data from the ZymoBIOMICS Microbial Community DNA Standard and compared the number of calls from the WIMP and 16S workflows at the genus (Fig. 3a) and species (Fig. 3b) levels. As expected for a quantitative workflow, the gDNA WIMP calls agree well with the theoretical levels. The identity calls from the 16S data correlate closely with those from the gDNA data, particularly at genus level, but the correct abundance of genera and species is not reflected in the 16S data, possibly due to PCR bias. This is most noticeable for Pseudomonas, to which our 16S primers had mismatches. At species level, the 16S data reveals some false positive calls, reflecting the similarity of the 16S sequences of some genera.

Fig. 4 DNA extraction, followed by CO1 PCR and library prep on VolTRAX

Sample to result: identification of insect species by CO1 sequencing using VolTRAX

In some situations it is an advantage to be able to identify species outside of a laboratory environment. VolTRAX is a portable device which is designed to perform the necessary steps to convert a raw biological sample to a form ready for analysis on a nanopore sensing device, without the need for human intervention. We extracted DNA from the invasive ladybird species Harmonia axyridis by bead-beating, and loaded the crude extract onto VolTRAX. We performed PCR of a 650 bp region of the cytochrome oxidase gene followed by addition of sequencing adapters using our rapid-attachment chemistry, on VolTRAX, and sequenced the resulting library for 1 hour (Fig. 4a). BLAST analysis of the reads confirmed the identity of the sample (Fig. 4b).

DNA sequencing in microgravity on the International Space Station (ISS) using the MinION

The portability, ease-of-use and low power requirements of Oxford Nanopore’s MinION enables sequencing in non-laboratory environments and even in microgravity!

Fig. 1 Astronaut Kate Rubins on the ISS

The first ever sequencing in space was performed on the ISS by Kate Rubins

The portability of the MinION allows users to sequence in non-laboratory situations. To demonstrate this, in collaboration with NASA, we tested the MinION aboard the ISS. The ISS orbits 400 km above the Earth, and travels at approximately 28,000 km/h and so is in constant freefall, with a continuous microgravity environment (Fig. 1). A mixture of lambda phage, mouse and E. coli DNA libraries was prepared on Earth and was sequenced on the ISS over four MinION runs. The same mixture of libraries was sequenced over four MinION runs on Earth, to act as a control. 

Fig.2 Analysis workflow showing read quality for runs a) on Earth b) on the ISS

Results from the first ever space sequencing done on MinION and R7.3 flow cells

We created a chained workflow consisting of 1D basecalling of raw fast5 files, 2D basecalling, extraction of quality scores and read-length information (Figs. 2a and 2b), and finally alignment. The workflow is capable of processing individual reads as soon as they are generated on the MinION, meaning that data can be analysed almost in real time. Due to internet limitations on the ISS, data was downloaded and processed immediately on Earth following completion of each run. In this way, basecalling and alignment of the data were performed almost simultaneously, allowing the success of the experiment to be confirmed shortly after the workflow was started.

Fig. 3 Percentage of reads mapping to each reference genome for Earth and ISS runs

Sequence data from ISS runs is indistinguishable from ground controls

For alignment, the workflow took 2D reads and used Minimap to establish whether each read mapped to E. coli K-12, lambda phage or mouse BALB/C genomes. Reads aligning to both lambda and E. coli genomes were resolved using BLAST to identify the correct placement. Any reads that could be resolved in this way were placed into the ‘Unknown’ group. Reads that did not align to any of the three reference genomes were placed into the ‘No_match’ group. Fig. 3  shows the percentage of reads assigned to these categories for three Earth and all four ISS runs together. Ground 2 was not included because the run was not successful. 

Fig. 4 Identification of bacterial genera found on Aquarius

NASA Extreme Environment Mission Operations (NEEMO) project

The NEEMO project involves performing scientific research in the Aquarius underwater laboratory, 5.6 km off the coast of Key Largo, Florida. The laboratory is 19 metres below the water surface, and the hostile environment is analogous to that found in space. We have been working with NASA to perform metagenomic surveys of various surfaces found on Aquarius. The approach involves DNA extraction, 16s PCR, and MinION sequencing to identify bacteria down to genus level (Fig. 4). In the future, similar surveys may be done on the ISS to monitor the astronauts’ environment, to check the cleanliness of the air and water, or to monitor changes in astronauts’ health by analysing their microbiomes or gene-expression levels. 

Hybrid assembly pipeline released (using Canu, racon and Pilon)

The long sequencing reads produced by Oxford Nanopore’s platforms enable the assembly of genomes with superior contiguity compared to those produced by second generation technologies.

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The megabase-sized fungal genome of Rhizoctonia solani assembled from nanopore reads only.

The ability to quickly obtain accurate genome sequences of eukaryotic pathogens at low costs provides a tremendous opportunity to identify novel targets for therapeutics, develop pesticides with increased target specificity and breed for resistance in food crops.

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TP53 gene mutation analysis in chronic lymphocytic leukemia by nanopore MinION sequencing

Background

The assessment of TP53 mutational status is becoming a routine clinical practice for chronic lymphocytic leukemia patients (CLL).

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Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing

Objectives: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity.

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Using MinION nanopore sequencing to generate a de novo eukaryotic draft genome: preliminary physiological and genomic description of the extremophilic red alga Galdieria sulphuraria strain SAG 107.79

Reported here is the de novo assembly of a eukaryotic genome using only MinION nanopore DNA sequence data by examining a novel Galdieria sulphuraria genome: strain SAG 107.79.

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Real-time detection of antibiotic-resistance genes using Oxford Nanopore Technologies’ MinION

“ARMA”: an analysis workflow for identification of antibiotic-resistant microorganisms in real time, with the potential for point-of-care use

Fig.1 Penicillin resistance in S. pneumoniae and antibiotic use

Antibiotic resistance increases with antibiotic use

Antibiotics and other antimicrobial agents are becoming less and less effective as microorganisms develop resistance to them. This is largely the result of the over-use of these medications over the past few decades (Fig. 1). Antimicrobial resistance is seen as one of the greatest threats to patients’ safety across the world.

Some bacteria are naturally resistant to certain classes of antibiotics, but acquired resistance is the more significant problem, and results from both new mutations and gene transfer between organisms.

Fig.2 VanA-type vancomycin-resistant Staphylococcus aureus

Inter-species gene transfer spreads antibiotic resistance

Horizontal gene transfer is considered to be the major cause of antibiotic resistance in bacteria. Here, genes that are responsible for resistance to one or more antibiotics in one species of bacterium can be transferred to other species by a variety of mechanisms. When another bacterium receives the genes, it acquires resistance to those antibiotics (Fig. 2). With this in mind, we sought to write an analysis workflow that could be run in real time in the cloud, which could take sequence reads generated from a bacterial sample and which would identify any antibiotic-resistance genes present, in real time.

Fig.3 Antibiotic resistance analysis report

Analysis workflow for antibiotic resistance allows real-time detection of resistance genes

We obtained resistance gene sequences and antibiotic-resistance ontology (ARO) from the Comprehensive Antibiotic Resistance Database (CARD, http://arpcard.mcmaster.ca/). The ARO describes how the genes are related to antibiotic drugs. During nanopore sequencing, as soon as a library strand passes through the nanopore, the data is available for basecalling. This allows us to perform analyses on individual reads in real time. The antibiotic-resistance workflow runs Oxford Nanopore Technologies’ standard 1D basecalling, and then uses lastal to align the 1D basecalls against the full set of antibiotic-resistance genes in the CARD database. The report highlights which alignments indicate resistance to a given antibiotic. Because many sequences in the resistance database are either duplicates or are closely related, we can expect identical or very similar alignments of reads against members of a given cluster (Fig. 3).

PCR-based, PCR-free, and rapid barcoding for nanopore sequencing libraries

PCR-based and PCR-free barcoding strategies for nanopore sequencing libraries, with 96 verified barcodes, and sequential loading of sequencing libraries onto a single reusable flow cell.

Fig. 1 PCR-based, PCR-free and rapid barcoding workflows, shown for gDNA templates

PCR-based and PCR-free barcoding for amplicons, cDNA or genomic DNA

Oxford Nanopore’s barcoding kits allow users to pool and sequence multiple libraries or amplicons in a single sequencing run, making more efficient use of the run. Barcoded libraries can then be pooled prior to sequencing, or libraries can be loaded sequentially onto the flowcell. There are currently three versions of the kit available, in which barcodes are added to ligation-based libraries by PCR or ligation, and to rapid libraries as part of the transposition reaction (Figs. 1a, 1b and 1c, respectively). We have also recently released a 4-primer PCR protocol which allows users to barcode their own locus-specific PCRs and to attach adapters using our rapid attachment chemistry.

Fig. 2 a) 96-plex barcoding of E. coli amplicons b) and c) sequential loading of libraries

Sequential loading of barcoded libraries onto a single flowcell, using our wash kit

96 PCR barcodes are currently available. These barcodes segregate cleanly (Fig. 2a), with 90% of reads having a barcode identified, and 98.8% of those reads having the correct barcode called. We are also increasing the number of available PCR-free ‘native’ barcodes from 12 to 24. Nanopore data is generated in real time, and runs can be stopped as soon as sufficient data has been obtained. A different library can then be loaded onto the same flowcell. However, strands from the previous library may still remain (Fig. 2b). Our wash kit removes the previous library, and cross-contamination is avoided by barcoding (Fig. 2c). The flowcell can be stored after washing, which is useful for when not all samples are available for library preparation at the start of a run.

Fig. 3 Four-primer barcoding a) workflow b) Bioanalyzer trace c) CO1 sequence alignment

Four-primer PCR for rapid PCR barcoding and attachment of sequencing adapters

To attach barcodes to locus-specific amplicons, our previous protocol required two consecutive PCR reactions. We have shortened this protocol substantially (Fig. 3a). The new protocol relies on a single nested PCR: locus-specific primers are used which are tailed with barcodes and universal sequences. Rapid attachment universal primers are also included in the PCR, resulting in tailed amplicons to which rapid adapters can be attached. Only successfully amplified nested PCR products are capable of receiving sequencing adapters. This final step takes approximately 10 minutes (Fig. 3b). Here we show results of performing this protocol on the human CO1 gene, with nanopore reads aligned to the NCBI reference (Fig. 3c).

Fig. 4 Dual barcoding a) workflow for PCR libraries b) test case c) results after splitting barcodes

Dual barcoding allows greater numbers of libraries to be pooled in a sequencing run

When looking at PCR amplicons, or plasmids, only a small amount of data is needed. Here, it would be helpful to be able to sequence larger numbers of templates on a single flowcell. To accommodate this, we are developing PCR-based (Fig. 4a) and PCR-free dual barcoding protocols. We tested the concept by taking swabs from a variety of environments and growing bacteria from each environment on a separate agar plate (Fig. 4b). We performed 16S PCR on each colony, and barcoded each set of amplicons with an inner, colony-specific barcode. We then made a pool of amplicons for each plate and labelled each pool with an outer, plate-specific barcode. Fig. 4c shows the counts of each barcode combination after splitting.

Extreme metagenomics using nanopore DNA sequencing: a field report from Svalbard, 78 N

In the field of observation, chance favours only the prepared mind (Pasteur). Impressive developments in genomics have led microbiology to its third ″Golden Age″.

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Enrichment by hybridisation of long DNA fragments for Nanopore sequencing

Enrichment of DNA by hybridisation is an important tool which enables users to gather target-focused next-generation sequence data in an economical fashion.

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Comparison of bacterial genome assembly software for MinION data and their applicability to medical microbiology

Translating the Oxford Nanopore MinION sequencing technology into medical microbiology requires on-going analysis that keeps pace with technological improvements to the instrument and release of associated analysis software.

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Benchmarking of de novo assembly algorithms for Nanopore data reveals optimal performance of OLC approaches.

Background

Improved DNA sequencing methods have transformed the field of genomics over the last decade.

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Highly parallel direct RNA sequencing on an array of nanopores

Ribonucleic acid sequencing can allow us to monitor the RNAs present in a sample.

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On the study of microbial transcriptomes using second- and third-generation sequencing technologies

Second-generation sequencing technologies transformed the study of microbial transcriptomes. They helped reveal the transcription start sites and antisense transcripts of microbial species, improving the microbial genome annotation.

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Rapid resistome mapping using nanopore sequencing

The emergence of antibiotic resistance in human pathogens has become a major threat to modern medicine and in particular hospitalized patients.

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de novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer

Background: Oxford Nanopore Technologies Ltd (Oxford, UK) have recently commercialized MinION, a small single-molecule nanopore sequencer, that offers the possibility of sequencing long DNA fragments from small genomes in a matter of seconds.

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What’s in my Pot? (WIMP), a quantitative analysis tool for real-time species identification

WIMP is a quantitative analysis tool for real-time species identification for bacteria, fungi, archaea and viruses. We apply it here to DNA extracted from soil around the roots of a Basmati rice plant

Fig. 1 WIMP report, shown for a sample containing bacteria, viruses and fungi

The WIMP application for bacterial, viral, fungal and archaeal species identification

The WIMP workflow classifies and identifies species in real time: as soon as a strand of DNA passes through the pore it can be basecalled and analysed. WIMP makes use of Centrifuge, which is capable of accurately identifying reads when using databases containing multiple highly similar reference genomes, such as different strains of a bacterial species. Centrifuge works by  identifying unique segments of those genomes and building an FM-index that can be used for efficient searches of sequenced reads. WIMP processes Centrifuge results to determine the most reliable placement in the taxonomy tree, assigning a score to each taxonomic placement. WIMP currently supports bacteria, archaea, viruses and fungi (Fig. 1).
 

Fig. 2 WIMP species quantification a) vs. qPCR b) on the Zymo microbial community standard

WIMP species counts correlate with qPCR and microbial standard quantification

To demonstrate that the WIMP workflow (including sequencing and library preparation) is quantitative, we took a selection of bacterial genomes, prepared sequencing libraries and pooled them in arbitrary ratios. We then compared the WIMP counts to relative abundance as measured by qPCR. The qPCR counts agreed well with the WIMP data (Fig. 2a). Next, we compared the WIMP counts on a microbial community standard to the manufacturer’s theoretical values and again saw good concordance (Fig. 2b). As a consequence, the limit of sensitivity of the workflow is essentially a question of how many reads are obtained from the sequencing run, which is governed by how long the device is left to generate data.

Fig. 3 Analysis of microbiome from soil around the roots of Basmati rice, shown at genus level

WIMP analysis of complex soil microbial community from Basmati rice roots

To demonstrate the effectiveness of WIMP for the analysis of a highly complex microbial community, we took a sample of soil from around the roots of a Basmati rice plant and extracted total genomic DNA by bead-beating. We prepared and sequenced an LSK-108 library and identified the organisms present with WIMP. The microbial community consisted largely of bacteria, though some fungal species were also identified (Fig. 3). WIMP identified many species of nitrogen-fixing bacteria, such as those belonging to the Bradyrhizobium and Rhizobium genera. Notably, WIMP also revealed the presence of several phytopathogens, including Xanthomonas translucens, which causes leaf streak, and can reduce crop yields substantially.    

Fig. 4 Analysis of rice soil microbes with 1D and 1D2 reads a) classification b) identified species

Reproducible species identification from 1D and 1D2 reads

We prepared 1D and 1D2 libraries from the rice soil metagenomic sample, to investigate the effect of using higher-accuracy reads on our ability to classify to species level. The results showed that approximately twice as many 1D2 reads could be classified by WIMP, compared to 1D reads (Fig. 4a). This is presumably due to a higher proportion of 1D2 reads passing WIMP’s in-built quality-score filtering. We compared the species identified from several 1D libraries with the 1D2 results, by exporting CSV files from WIMP, and by generating heat-maps from the data (Fig. 4b). The results from all libraries were in very close agreement, indicating that it is not essential to use the higher-accuracy 1D2 reads for robust species identification with WIMP. 

Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing

Demands for faster and more accurate methods to analyze microbial communities from natural and clinical samples have been increasing in the medical and healthcare industry.

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De novo assembly of prokaryotic and large eukaryotic genomes with long nanopore reads

The long-read capability of nanopore sequencing simplifies de novo assembly of microbial and eukaryotic whole genomes, resulting in increased assembly contiguity

Fig. 1 Illustration of a typical approach to genome assembly

Long nanopore reads enable de novo assembly of large and complex genomes

Nanopore reads can reach hundreds of kilobases in length, which is more than sufficient to span entire viral genomes in single reads. In contrast, to obtain a complete genome sequence from bacterial, or larger, genomes it is currently necessary to reconstruct the sequence by aligning and joining together overlapping sequence reads. This process is termed ‘de novo genome assembly’ (Fig. 1). Assembling genomes using data from short-read sequencing technologies presents a computational challenge, and the results tend to be imperfect, particularly when the genomes contain extensive repetitive regions. Long reads make assembly far easier, and allow us to resolve repeats and structural variants that are several kilobases in length.

Fig. 2 Longer reads allow complete assembly from lower-coverage data

Long reads allow the complete assembly of genomes at lower coverage

To explore the influence of read length and depth of coverage on genome assembly we generated an excess of 1D sequence data for the E. coli K-12 strain. We filtered the data by minimum length and by the number of bases, and assembled the resulting FASTQ files using miniasm (Fig. 2a). We counted the number of contigs produced, as a measure of the completeness of the assemblies. Our results show that the longer the reads used, the lower the coverage needed to achieve a complete, single-contig assembly (Fig. 2b). Fig. 2c shows a mummer plot of the fully assembled genome obtained from 20x coverage of reads greater than 20 kb, against the K-12 reference. 

Fig. 3 Comparison of assembly completeness and identity for 1D and 1D2 reads

Higher single-read accuracy of 1D2 reads improves contig length and assembly quality

To investigate the effect of higher single-read accuracy reads on the contiguity and accuracy of de novo assembly, we sequenced PCR-free 1D and 1D2 libraries from E. coli genomic DNA. We assembled the resulting data with Canu, using reads > 5 kb (Fig. 3a) and > 20 kb (Fig. 3b). We obtained longer contigs from lower-coverage 1D2 reads compared to 1D reads, with a less pronounced effect for the dataset containing only reads > 20 kb. When Racon was used to polish the assemblies, a higher identity was obtained from the 1D2 assembly compared to 1D  (Fig. 3c), though this difference was reduced when the assemblies were polished using a combination of Racon and methylation-aware nanopolish (Fig. 3d). The software tools used to calculate identity are available at https://github.com/nanoporetech/wub

Fig. 4 Basmati rice assembly a) Mummer plot against reference genome b) contig length

Assembly of aromatic Basmati rice with long nanopore reads

Basmati rice originated in the foothills of the Himalayas, and has become a highly valuable crop due to its distinctive aroma and slender grain morphology. As part of a larger ongoing collaboration to compare the genome structures of two different Basmati strains, we extracted gDNA from leaves of the Indian strain, Basmati 334, and prepared sequencing libraries both with and without shearing. We ran these libraries on a MinIONTM and generated approximately 1.6 Gb of 1D sequence data. We assembled the reads using miniasm and then polished the assembly using Racon. The resulting assembly consisted of 844 contigs (Fig. 4a). with an N50 of 1,366,165 bp, and a total assembly size of 383,222,097 (Fig. 4b). We are also in the process of assembling the data using Canu.

Potential for self-monitoring of chronic myelogenous leukaemia gene fusion using VolTRAX and MinION

Quantitative home-monitoring of the BCR-ABL1-expressing Philadelphia chromosome by automated library preparation on VolTRAX, sequencing on MinION and analysis.

Fig. 1 Schematic representation of the BCR and ABL1 genes that break and fuse

Overview of CML: BCR-ABL1 fusion gene arises from Philadelphia translocation

The Philadelphia chromosome is a rearrangement that is present in the white blood cells of 90% of people with chronic myelogenous leukaemia (CML). It arises from a chromosome 9 and chromosome 22 translocation, generating a fusion gene from the breakpoint cluster region (BCR) and the Abelson leukaemia (ABL1) gene. Several breakpoints have been identified in BCR, and the fusion of these different breakpoints to ABL1 results in the production of a non-regulated tyrosine kinase, transforming normal cells to neoplastic CML cells, and leading to unlimited propagation. BCR exon 13–ABL1 exon 2 (e13a2, p210) and BCR exon 14–ABL1 exon 2 (e14a2, p210) have been found in more than 95% of CML patients (Fig. 1). 

Fig. 2 Alignment of MinION reads to ABL1, BCR and BCR_ABL1 reference sequences

Detecting BCR-ABL1 fusion and ABL1 2D cDNA amplicons on MinION

MinION sequencing can be used to detect the presence of the BCR-ABL1 fusion transcript, and by using the normal ABL1 transcript as an endogenous control we have developed a quantitative assay. After mRNA extraction and cDNA synthesis, we run two gene-specific PCR reactions, each for a limited number of cycles, one amplifying ABL1, and the other amplifying BCR-ABL1. The BCR-ABL1 primers are positioned in such a way that they amplify all fusion breakpoints, making use of the long-read capability of the MinION. Fig. 2 shows alignments of the amplicon sequence data back to the ABL1 and the BCR-ABL1 fusion gene reference sequences.

Fig. 3 Using MinION sequencing reads as a quantitative assay for BCR_ABL1

Quantitative monitoring of BCR-ABL1/ABL1 ratios by MinION sequencing

The expression level of the BCR-ABL1 fusion transcript is reduced by successful treatment, but relapses do occur, and sensitive and quantitative methods are required for their detection. RT- qPCR-based methods are commonly used to detect both the BCR-ABL1 fusion and ABL1 transcript, which is used as an endogenous control. In our sequencing-based workflow, the original BCR-ABL1/ABL1 ratio changes after the two gene-specific PCRs, due to different PCR protocols and efficiencies. Therefore, a correlating curve is built from a series of qPCR measurements, allowing conversion from the post-PCR ratio of BCR-ABL1/ABL1 to the pre- PCR ratio (Fig. 3).  

Fig. 4 Monitoring in a non-laboratory environment

MinION and VolTRAX allow testing in non- laboratory environments

The portability of the MinION sequencer, coupled with VolTRAX, our automated sample and library-preparation device, means that it is possible to perform sequencing-based assays outside of a laboratory environment. Together with user-friendly analyses, this could potentially allow testing for chronic conditions like CML to be performed in a doctor’s surgery. As well as giving greater control to the patient, and making the testing process more convenient, this approach could minimise the amount of time taken to return results, meaning that any necessary action could be taken within hours of taking the sample, in contrast to the current waiting time of several weeks.

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%.

Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

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.

Quality Assessment Tools for Oxford Nanopore MinION data

IONiseR provides tools for the quality assessment of Oxford Nanopore MinION data. It extracts summary statistics from a set of fast5 files and can be used either before or after base calling.

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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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De novo sequencing and variant calling with nanopores using PoreSeq

The accuracy of sequencing single DNA molecules with nanopores is continually improving, but de novo genome sequencing and assembly using only nanopore data remain challenging.

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LAST

Martin Frith, Computational Biology Research Center in Tokyo

Release Date: 18-Sep-2015

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Fast genome and metagenome distance estimation using MinHash

Given a massive collection of sequences, it is infeasible to perform pairwise alignment for basic tasks like sequence clustering and search.

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Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences

Motivation: Single Molecule Real-Time (SMRT) sequencing technology and Oxford Nanopore technologies (ONT) produce reads over 10kbp in length, which have enabled high-quality genome assembly at an affordable cost.

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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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BioMed Central blog: Real-time surveillance with nanopore-seq

Rafal Marszalek at Genome Biology notes that “Lessons about the epidemic control are usually learned the hard way.

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Josh Quick: Lab in a suitcase and other adventures with Nanopore sequencing

Josh Quick describes his recent work to monitor ebola in Guinea using the MinION.

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New Zealand Herald on MinION for mitochondrial genomes and pathogen monitoring

The New Zealand Herald interviews David Eccles on his work using MinION: “The device has been specifically used by the institute to sequence and com

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Surveillance of Ebola in Guinea using the MinION – the story

Nick Loman blogs about his group’s participation in the monitoring of Ebola in Guinea, describing the work of Josh Quick in the field.

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MinION in space

We are delighted that the MinION is scheduled to visit the International Space Station; this article from NASA explains what the project aims to achie

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A novel method for the multiplexed target enrichment of MinION next generation sequencing libraries using PCR-generated baits

The enrichment of targeted regions within complex next generation sequencing libraries commonly uses biotinylated baits to capture the desired sequences. This method results in high read coverage over the targets and their flanking regions.

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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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npReader – real-time conversion and analysis of Nanopore reads

npReader (jsa.np.f5reader) is a program that extracts Oxford Nanopore sequencing data from FAST5 files, performs an initial analysis of the date and streams them to real-time analysis pipelines.

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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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ZiBRA project: real-time sequencing of Zika virus in Brazil

A revolution is occurring in genomic epidemiology. Recently, real-time portable genome sequencing using the Oxford Nanopore MinION device was successfully used to characterize the genetic diversity of the Ebola virus outbreak in Guinea.

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Sequencers for Soldiers: Battlefield Genomics

Claire Lonsdale from the Defence Science and Technology Laboratory talks to the Nanopore community at London Calling 2016.

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Sara Goodwin, CSHL: Rapid CNV Characterisation of clinical cancer samples on the Oxford Nanopore MinION

Sara Goodwin of Coldspring Harbour Laboratory talks to the Oxford Nanopore Community at London Calling.

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Clive Brown, CTO of Oxford Nanopore, talks at the London Calling conference

Clive is Chief Technology Officer at Oxford Nanopore. On the Executive team, he is responsible for all of the Company’s product-development activities.

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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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Applications of Nanopore Sequencing for Infectious Disease Detection

A talk by Stephanie Hao of Johns Hopkins University Applications of Nanopore Sequencing for Infectious Disease Detection 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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How MinION is Changing my Research in Infectious Diseases Diagnostics

A presentation to the MinION Community by Dr Justin O’Grady, Lecturer in Medical Microbiology, University of East Anglia.

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Rapid Short-Read Sequencing and Aneuploidy Detection using MinION Nanopore Technology

A presentation to the MinION Community by Dr Zev Williams, Assistant Professor, Department of Obstetrics & Gynecology and Women’s Health, Assistant Professor, Department of Genetics, Albert Einstein College of Medicine.

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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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Running and Reading in Real Time: Looking at Squiggles on the MinION

Dr Matthew Loose, Head of Next Generation Sequencing Service, Nottingham University talks to the MinION commnity.

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Getting the Flu: Exploring Influenza Virus Evolutionary Dynamics by Single Molecule Sequencing

Elodie Ghedin - Professor of Biology and member of the Center for Genomics and Systems Biology, New York University - talks to the MinION community.

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Leveraging MinHash for Rapid Identification of Nanopore Data on Mobile Hardware

Dr Brian Ondov, Bioinformatics Engineer at National Biodefense Analysis and Countermeasures Centre talks to the MinION Community about Leveraging MinHash.

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NGS for Public Health Microbiology

Dr Catherine Arnold, Consultant Clinical Scientist in Molecular Microbiology at Public Health England talks to the MinION Community about NGS for Public Health Microbiology.

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Real-time Genomic Characterization of Viral Threat Agents using Nanopore Sequencing

Dr Andrew Kilianski presents a talk for the MinION Community on Real-time Genomic Characterization of Viral Threat Agents using Nanopore Sequencing.

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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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A year of happy MAPping

In this talk I will cover the highs and lows of being part of the Oxford Nanopore MinION Access Programme. Our laboratory joined the MAP programme in May 2014.

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Nanopore sequencing – disruptive technology in clinical microbiology?

The diagnosis of infectious diseases by culture takes at least two days: one to grow the bacteria and then, at best, one to identify pathogens and test their antimicrobial susceptibility. Meanwhile the patient is treated empirically, which often results in inappropriate treatment.

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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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Whole genome sequencing of influenza virus genomes using MinION

A presentation by Nicole Moore at London Calling 2015.

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Real-time identification of pathogens and antibiotic resistance profile using Oxford Nanopore sequencing

Clinical pathogen sequencing has been demonstrated to have a positive outcome on treatment of patients with unknown bacterial infection. However, widespread adoption of clinical pathogen sequencing has been impeded by the lack of real-time sequencing devices.

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minoTour – real time analysis tools for minIONs

Nanopore sequencing introduces true real-time sequencing for the first time. Full exploitation of real-time sequencing requires a novel approach to data analysis for which we have developed the minoTour platform.

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Sequence capture on the MinION

A presentation at London Calling 2015 by Daniel Fordham.

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Modified base detection using Oxford Nanopore MinION (London Calling)

A presentation at London Calling 2015 by Rachael Workman.

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Direct RNA Sequencing

Libby Snell, Oxford Nanopore Technologies

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Error correction, assembly and consensus algorithms for MinION data

In my talk, I will discuss my collaboration with Nick Loman’s lab to develop de novo assembly methods for MinION data. We have built a pipeline to error correct nanopore reads using partial order graphs and the corrected reads are subsequently assembled using the Celera Assembler.

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Democratizing DNA Fingerprinting

We report a rapid, inexpensive, and portable strategy to re-identify human DNA using the MinION, a miniature sequencing sensor by Oxford Nanopore Technologies. Our strategy requires only 10-30 minutes of MinION sequencing, works with low input DNA, and enables familial searches.

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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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Comparison of bacterial genome assembly software for MinION data

Antimicrobial resistance genes can be carried on plasmids or on mobile elements integrated into the chromosome.

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Centrifuge: rapid and sensitive classification of metagenomic sequences

Centrifuge is a novel microbial classification engine that enables rapid, accurate and sensitive labeling of reads and quantification of species on desktop computers.

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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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Use of Unamplified RNA/cDNA–Hybrid Nanopore Sequencing for Rapid Detection and Characterization of RNA Viruses

Nanopore sequencing, a novel genomics technology, has potential applications for routine biosurveillance, clinical diagnosis, and outbreak investigation of virus infections.

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Enrichment of long DNA fragments from mixed samples for Nanopore sequencing

Whole-genome sequencing of pathogenic organisms directly from clinical samples combines detection and genotyping in one step.

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Integration of mobile sequencers in an academic classroom

The advent of mobile DNA sequencers has made it possible to generate DNA sequencing data outside of laboratories and genome centres. Here, we report our experience of using the MinION, a mobile sequencer, in a 13-week academic course for undergraduate and graduate students.

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Cytosine Variant Calling with High-throughput Nanopore Sequencing

Chemical modifications to DNA regulate cellular state and function. The Oxford Nanopore MinION is a portable single-molecule DNA sequencer that can sequence long fragments of genomic DNA.

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DeepNano: Deep Recurrent Neural Networks for Base Calling in MinION Nanopore Reads

Motivation: 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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Nanocall: An Open Source Basecaller for Oxford Nanopore Sequencing Data

Motivation: The highly portable Oxford Nanopore MinION sequencer has enabled new applications of genome sequencing directly in the field.

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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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Real time selective sequencing using nanopore technology

The Oxford Nanopore Technologies MinION sequencer enables the selection of specific DNA molecules for sequencing by reversing the driving voltage across individual nanopores. To directly select molecules for sequencing, we used dynamic time warping to match reads to reference sequences.

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Real-time, portable genome sequencing for Ebola surveillance.

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths. Genome sequencing in viral outbreaks is desirable to characterise the infectious agent and determine its evolutionary rate.

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Species level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer

Background: The miniaturised and portable DNA sequencer MinION has been released to the scientific community within the framework of an early access programme to evaluate its application for a wide variety of genetic approaches.

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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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Rapid short-read sequencing and aneuploidy detection using MinION nanopore technology

MinION™ is a memory stick-sized, nanopore-based sequencer primarily designed for single-molecule sequencing of long DNA fragments (>6 kb).

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Rapid antibiotic resistance predictions from genome sequence data for S. aureus and M. tuberculosis

Rapid and accurate detection of antibiotic resistance in pathogens is an urgent need, affecting both patient care and population-scale control. Microbial genome sequencing promises much, but many barriers exist to its routine deployment.

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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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Nanopore Sequencing in Microgravity

The ability to perform remote, in situ sequencing and diagnostics has been a long-sought goal for point-of-care medicine and portable DNA/RNA measurements.

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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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Complete nitrification by Nitrospira bacteria

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be a two-step process catalysed by chemolithoautotrophic microorganisms oxidising either ammonia or nitrite.

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Snake venom gland cDNA sequencing using the Oxford Nanopore MinION portable DNA sequencer

Portable DNA sequencers such as the Oxford Nanopore MinION device have the potential to be truly disruptive technologies, facilitating new approaches and analyses and, in some cases, taking sequencing out of the lab and into the field.

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Draft Genome Sequence of the Pandoraea apista LMG 16407 Type Strain

Pandoraea species, in particular Pandoraea apista, are opportunistic, multidrug-resistant pathogens in persons with cystic fibrosis (CF).

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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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Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

We report unbiased metagenomic detection of chikungunya virus (CHIKV), Ebola virus (EBOV), and hepatitis C virus (HCV) from four human blood samples by MinION nanopore sequencing coupled to a newly developed, web-based pipeline for real-time bioinformatics analysis on a computational server or la

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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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Noninvasive Prenatal Testing by Nanopore Sequencing of Maternal Plasma DNA: Feasibility Assessment

Noninvasive prenatal testing (NIPT) by maternal plasma DNA sequencing is now clinically available for screening fetal chromosomal aneuploidies; these tests have close to 99% sensitivity..

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MinION nanopore sequencing of an influenza genome

Influenza epidemics and pandemics have significant impacts on economies, morbidity and mortality worldwide. The ability to rapidly, and accurately, sequence influenza viruses is instrumental in the prevention and mitigation of influenza.

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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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Real-time digital pathogen surveillance — the time is now

It is time to shake up public health surveillance. New technologies for sequencing, aided by friction-free approaches to data sharing, could have an impact on public health efforts.

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Early insights into the potential of the Oxford Nanopore MinION for the detection of antimicrobial resistance genes

Genome sequencing will be increasingly used in the clinical setting to tailor antimicrobial prescribing and inform infection control outbreaks. A recent technological innovation that could reduce the delay between pathogen sampling and data generation is single molecule sequencing.

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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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A complete bacterial genome assembled de novo using only nanopore sequencing data

We have assembled de novo the Escherichia coli K-12 MG1655 chromosome in a single 4.6-Mb contig using only nanopore data.

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Nanopore sequencing of ebola viruses under outbreak conditions

Determining the full-length genome sequences of viruses during disease outbreaks such as the ongoing Ebola virus outbreak in West Africa, which is of unprecedented scale with about 24,000 cases and 10,000 deaths as of March 2015, can provide important information about virus evolution, and ensure

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MinIONs and Nanofrogs

Until today, the investigation of the biological diversity of many parts of the world has been obstructed by the inability to perform field based DNA analysis, especially in those regions where the biological richness is higher, like the countries in the inter-tropical area.

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Community utility of nanopore data | Ewan Birney

Ewan Birney gives a talk at London Calling 2015 on community utility of nanopore data.

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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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Identifying biological samples with the MinION: scientific challenges and potential applications

Dr. Brook Milligan is Director of the Conservation Genomics Laboratory and Professor of Biology at New Mexico State University. After earning a B.A. in physics from Dartmouth College and a Ph.D. in ecology from the University of California, Davis, Dr.

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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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Nanopore sequencing for genotyping pathogens of tropical diseases

Nanopore sequencer, MinION, has enabled sequencing analysis without pre-installation of expensive conventional sequencers or pre-requisite of specific skills in biological experiments. Even electric supply is not always necessary, by connecting MinION to a laptop PC.

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Nanopore-based 5D fingerprinting of single proteins in real-time (London Calling Presentation)

Are 1-D and 2-D gel-electrophoresis the best we can do in routine protein analysis? This talk demonstrates that by moving proteins through nanopores, it is possible to separate these proteins transiently from other macromolecules in solution.

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The MinION: Applications in Food safety (London Calling presentation)

Nanopore sequencing represents a paradigm shift in DNA sequencing, and today it is the only sequencing technology that measures an actual single molecule of DNA.

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Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella

Foodborne outbreaks of Salmonella remain a pressing public health concern. We recently detected a large outbreak of Salmonella enterica serovar Enteritidis phage type 14b affecting more than 30 patients in our hospital.

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Yaniv Erlich: A vision for Ubiquitous Sequencing

Yaniv Erlich outlines his vision for a future where “the next goal of the revolution can be ushered in by the advent of sequencing sensors – miniaturized sequencing devices that are manufactured for real time applications and deployed in large quantities at low costs.”

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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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Successful test launch for nanopore sequencing

Nanopore sequencing gets a boost with accurate error modelling and variant-calling tools for Oxford Nanopore Technology’s highly anticipated MinION platform.

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Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer

The MinION™ nanopore sequencer was recently released to a community of alpha-testers for evaluation using a variety of sequencing applications.

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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.

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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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marginAlign, marginCaller, marginStats – tools to align nanopore reads to a reference genome

The marginAlign package can be used to align reads to a reference genome and call single nucleotide variations (SNVs). It is specifically tailored for Oxford Nanopore Reads

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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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Mitochondrial Genome Acquisition Restores Respiratory Function and Tumorigenic Potential of Cancer Cells Without Mitochondrial DNA

We report that tumor cells without mitochondrial DNA (mtDNA) show delayed tumor growth, and that tumor formation is associated with acquisition of mtDNA from host cells.

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nanoCORR – error correction tool for nanopore sequence data

Sara Goodwin and James Gurtowski at Cold Spring Harbor Laboratory have created error correction software for Oxford Nanopore data

Release Date: 24-Aug-2015

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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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poRe: an R package for the visualization and analysis of nanopore sequencing data

Motivation: The Oxford Nanopore MinION device represents a unique sequencing technology. As a mobile sequencing device powered by the USB port of a laptop, the MinION has huge potential applications.

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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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Detecting DNA Methylation using the Oxford Nanopore Technologies MinION sequencer

Nanopore sequencing instruments measure the change in electric current caused by DNA transiting through the pore. In experimental and prototype nanopore sequencing devices it has been shown that the electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine.

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