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CLINICAL RESEARCH

The promise of nanopore sequencing for clinical and cancer research

OXFORD NANOPORE TECHNOLOGIES | THE PROMISE OF NANOPORE SEQUENCING FOR CLINICAL AND CANCER RESEARCH

1Advantages of nanopore sequencing for clinical research

2Case studies

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About Oxford Nanopore Technologies

References

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5

Summary

Contents

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The widespread implementation of traditional sequencing technologies over the last decade has delivered unprecedented insight into human health and disease. Providing high-resolution analysis of multiple genomic loci across many samples, sequencing has become the standard approach for many clinical research applications.

While such technologies have proven adept at detecting single nucleotide variants (SNVs) in many areas of the genome, the inherent reliance on short sequencing reads (150–300 bp) limits their ability to detect other important sources of genomic variation, such as structural variation (SV), repetitive regions, phasing, and transcript isoform expression. Furthermore, the expense, large size, and infrastructure requirements of these platforms has the potential to limit

their application to all but the most well-resourced settings. Such centralisation of technology could lead to inequitable global access to the benefits of DNA and RNA sequencing, and increase time to result for smaller local or resource-limited laboratories.

Delivering ultra-long reads (up to 2 Mb1), real-time results, and scalable throughput — from portable to benchtop devices — nanopore sequencing offers a cost-effective solution to the challenges faced by traditional sequencing platforms.

This review outlines the advantages of nanopore sequencing for the detection of a variety of genomic and epigenomic variants. Specific case studies reveal how clinical researchers are now utilising nanopore sequencing to deliver new insights into human health and disease.

Introduction

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Detect a greater range of genomic variation with a single technology

Sequencing technologies have traditionally relied on the fragmentation and subsequent amplification of short lengths of DNA or RNA, typically between 150 to 300 bp in length. During this process, the relative positional information of each fragment is lost and, for most applications, must be pieced back together through overlap with other short fragments — adding significant time and complexity

Advantages of nanopore sequencing for clinical research1

Figure 1Schematic highlighting the advantages of long, direct sequencing reads in the de novo assembly and characterisation of a wide variety of genomic variants, including SNVs (black nucleotides), repetitive regions (blue boxes) and base modifications (red nucleotide depicting 5-methylcytosine). Long sequencing reads allow complete resolution of the genomic region, including base modifications, for both maternal and paternal alleles, while short sequencing reads struggle to resolve genomic repeats and phasing, and require a separate sequencing run to identify base modifications. Dotted line indicates alignment gap between both alleles due to different number of repeats.

to analyses. While this may not be a significant issue when examining individual SNVs or mutation hotspots, it confounds the analysis of many other common forms of genomic variation, including SV, repeat regions, phasing, and transcript isoforms (Figure 1).

It is well established that some repeat regions, SVs, and RNA isoforms are associated with human disease (e.g. triplet expansion diseases2, autism3, epilepsy4, and cancer5,6,7), making their routine

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characterisation highly advantageous. In addition, the discovery of novel variations of these types is now a key focus for researchers investigating disease pathogenesis and for the identification of biomarkers that may predict treatment response, disease progression, or even represent a drug target.

With nanopore sequencing, read length is equal to DNA or RNA fragment length. Complete fragments of thousands of kilobases are routinely sequenced and ultra-long read lengths over two megabases have been achieved1. Clearly, such long reads are more likely to span entire regions of repetitive DNA and SVs (see Case studies 1, 3, and 4). Furthermore, long nanopore sequencing reads have been demonstrated to allow discrimination of highly related gene family members and pseudogenes9. As a result, nanopore sequencing provides a more complete view of genetic variation.

As researchers search for new disease associations, haplotype phasing (determining from which chromosome a section of DNA is derived) is of increasing interest. Phasing can also resolve compound heterozygosity, where both alleles of a specific gene or region possess a different variant (see Case study 4). The identification of such variants may have significant implications to the future development and administration of personalised treatment strategies10. The long and ultra-long reads provided by nanopore sequencing simplify the phasing of large tracts of DNA, as recently demonstrated by the phasing of the entire 4 Mb major histocompatibility complex (MHC)11. The typing of some MHC genes is a vital component of successful organ transplantation. The facility of nanopore sequencing to phase relevant MHC genes may allow researchers to discover additional markers of importance for enhanced transplantation success12,13.

‘SVs represent an important class of genetic variation that accounts for a far greater number of variable bases than single nucleotide variations (SNVs)’ 8.

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Figure 2Overview of typical CRISPR/Cas workflow. CRISPR/Cas-based enrichment provides simple and effective way of rapidly enriching for long native DNA regions of interest (ROI), while preserving base modifications.*

Importantly, the advantages of nanopore sequencing are not limited to whole genome or whole transcriptome analysis. The long sequencing reads delivered by nanopore technology also expand the capabilities of targeted sequencing, allowing routine analysis of a broader range of genomic variants. All common targeting and enrichment strategies can be utilised, including PCR amplification (amplicon), hybridisation-based capture and CRISPR/Cas-mediated enrichment, with the latter providing a highly streamlined workflow and maintenance of base modifications (Figure 2).*

The power of full-length RNA transcripts

The analysis of RNA transcripts allows the comparison of gene expression across different cells and at different disease states, with some transcripts (e.g. fusions) being indicative of specific diseases such as cancer. As a single gene can encode many different transcripts (isoforms), it is exceptionally challenging when using short sequencing reads to assemble complete and unambiguous transcript isoforms. It has been reported that automated transcript assembly methods fail to

‘By exploiting the long reads produced by MinION sequencing, it is possible to evaluate the phasing of mutations, clarifying the allelic context of mutations affecting the same gene, but too geographically distant to be detected with short reads sequencing’ 14.

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* Oxford Nanopore Technologies does not sell a kit that enables CRISPR/Cas-mediated enrichment. Use of this technique may require rights to third-party owned intellectual property.

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Figure 3Nine of the top ten most abundant CACNA1C isoforms identified in brain tissue were novel, highlighting the benefits of long reads for isoform characterisation. Image courtesy of Dr. Michael Clark, University of Oxford, UK16.

identify all constituent exons in over half of the transcripts analysed15. Nanopore technology resolves these issues by sequencing the entire, full-length RNA molecule. Using the MinION, researchers identified 90 transcript isoforms of the neuropsychiatric disease-associated gene CACNA1C, of which only 7 had been previously reported16. Interestingly, 9 of the top 10 expressed isoforms were novel, previously unknown transcripts (Figure 3). Researchers have also used nanopore sequencing to successfully detect novel isoforms using targeted RNA capture17 and single cell transcriptomics18 approaches.

‘We are working on identification of alternative gene isoforms and fusion genes in cancer, and the ability to generate reads that span the entire RNA can make a big difference’ 50.

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CACNA1C GENCODE v27TSS

Annotated isoform Novel coding isoform Novel noncoding isoform (predicted frameshift)

Oxford Nanopore’s recently updated RNA sequencing kits offer significantly higher throughput from lower sample amounts for more sensitive analyses (Table 1). Nanopore sequencing is also the only technology currently capable of directly sequencing native RNA, without the requirement for prior conversion to cDNA. Not only does this eliminate potential bias introduced by amplification or reverse transcription, but it also enables the detection of base modifications alongside nucleotide sequence. The streamlined protocol has also been used to rapidly characterise RNA viruses19. In addition, direct and PCR-based cDNA sequencing kits provide high-depth sequencing of full-length transcripts and allow accurate quantification and identification of transcript isoforms and fusion transcripts (see Case study 2).

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Direct RNA Sequencing Kit

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Input requirement 500 ng RNA poly-A+ 1 ng RNA poly-A+ (or 50 ng total RNA)

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1 million/8 million 7–12 million/>60 million 5–10 million/30–60 million

Multiplexing options In development Yes Yes

Table 1RNA sequencing kits available from Oxford Nanopore Technologies.

Identify epigenetic modifications alongside nucleotide sequence

Nucleotide variation does not fully explain disease susceptibility; an increasing number of human diseases are now known to be associated with base modifications (e.g. the addition of a methyl group to cytosine to form 5-methylcytosine [5mC]). Methylation plays a key role in gene expression and, as a result, methylated bases have been linked to many human diseases, including neurological disorders20 and cancer21, and may offer significant potential as a diagnostic and prognostic indicator.

The amplification of nucleic acids — a prerequisite for traditional short-read sequencing technologies — erases base modifications, meaning they cannot be detected without additional time-consuming and often inefficient sample processing methods such as bisulfite conversion5,22.

Nanopore sequencing does not require amplification or strand synthesis, allowingboth the base and its modification to be detected in the same sequencing run (see Case study 1). In conjunction with recently developed CRISPR/Cas9-based target enrichment methodologies, nanopore sequencing also enables direct analysis of modified bases in specific genomic regions of interest23.

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Recently, researchers investigating lower respiratory tract infections (LRI) described how nanopore sequencing provided accurate pathogen identification and associated antimicrobial resistance profiling within just 6 hours30. Not only is this a considerable time saving compared to existing methods, which take on average 48–72 hours, it can be performed without a priori information on the infectious agent and allows the identification of novel resistance mutations, which is not possible using traditional PCR-based techniques (see Case study 6).

Certain forms of cancer require and respond well to rapid implementation of the appropriate treatment regime. Recently, a number of researchers have described the use of nanopore sequencing to investigate gene fusion events indicative of different forms of leukaemia, with one team reporting confident identification of the PML-RARA fusion in less than 50 minutes of sequencing, with the first PML-RARA read being detected in just over 1 minute7,32,33. This compares

‘Nanopore sequencing allows same-day detection of structural variants, point mutations, and methylation profiling using a single device with negligible capital cost’ 5.

To date, researchers have utilised nanopore sequencing to detect a number of modified bases from both DNA and RNA, including 5-methylcytosine (5mC)11, 5-hydroxymethylcytosine (5hmC)24, pseudouridine25, N6-methyladenosine (m6A)26, and 7-methylguanosine (m7G)22.

Rapid, real-time results

Unlike other sequencing technologies, which deliver all data at the end of a fixed and usually lengthy run time, nanopore sequencing provides data in real-time, allowing immediate access to results. Using nanopore technology, accurate identification of pathogens such as Ebola27, dengue, chikungunya, Mycobacterium tuberculosis28, and Candida auris29 has been demonstrated within a few minutes of sequencing (Figure 4).

Dengue virusChikungunya virus

Time (seconds) Time (seconds)

Figure 4Proportion of chikungunya virus (CHIKV) and dengue virus (DENV) genomes sequenced in real-time from a human clinical research sample exhibiting co-infection with both viruses. Near complete coverage was achieved within 4 seconds and 8 minutes for CHIV and DENV respectively31.

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extremely favourably with the commonly used technique of fluorescence in situ hybridisation (FISH) which typically takes 24-48 hours (see Case study 2). Furthermore, successful gene fusion detection was also achieved from formalin-fixed, paraffin-embedded (FFPE) sarcoma samples, which typically exhibit significantly lower DNA and RNA quality7.

In addition to offering rapid detection, real-time sequencing allows researchers to stop sequencing once a result has been obtained, wash, and then reuse the flow cell — providing additional cost savings to alternative sequencing-based approaches.

Cost-effective, scalable, and on-demand analysis

The unique capabilities of nanopore technology make it suitable for a wide range of potential assays, allowing the detection of SNPs, SVs, repeats, phasing, gene fusions, and full-length transcript isoforms. A range of devices are available to suit all throughput requirements (Figures 5–8).

Figure 5 Flongle: a flow cell adapter for MinION and GridION, providing up to 1.8 Gb of data for smaller, more frequently performed analyses. Each flow cell provides 126 channels.

Figure 6MinION: a powerful, portable device capable of delivering up to 30 Gb of data.

Figure 7GridION X5: 5 independent flow cells with integrated data processing.

Figure 8PromethION P24 and P48: 24 or 48 independent flow cells delivering up to 4.8 and 9.6 Tb of data respectively. Integrated data processing for high-throughput sequencing.

The MinION™ Starter Pack costs just $1,000 (including two flow cells and sequencing reagents) and delivers up to 30 Gb data per flow cell. GridION™ X5, PromethION™ 24, and PromethION 48 offer respectively 5, 150, and 300 times the yield of the MinION, providing users with the facility to cost-effectively scale their analyses to meet their laboratory’s specific requirements. These devices are available with no capital expenditure and deliver a comparable cost-per-base to traditional sequencing platforms. In addition, the facility to use flow cells independently allows a variety of assays to be run concurrently with no need for sample batching — delivering faster access to results.

For smaller, more frequently performed analyses, Oxford Nanopore offers Flongle, a low-cost flow cell adapter for MinION and GridION, which, in initial customer testing, delivers up to 1.8 Gb of data.

A range of streamlined library preparation kits are available offering the facility for sample multiplexing to further increase throughput and reduce costs.

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Accessible to any laboratory

Combining high performance with low cost and small size, nanopore devices ensure that the advantages of high-throughput sequencing are no longer limited to well-funded centralised service laboratories. The pocket-sized MinION is uniquely portable making it ideal for local implementation and resource-limited settings that ordinarily might not have access to such powerful and versatile sequencing technology (see Case study 6). Data analysis can be performed on a standard laptop or using MinIT™, a small yet powerful data-analysis accessory (Figure 9). Oxford Nanopore has released a growing number of real-time data analysis tools covering a range of routine applications, such as species identification and antimicrobial profiling. Sequencing data can also be exported in standard FASTQ and FAST5 file format suitable for further downstream analysis using a wide range of commonly used tools.

The small footprint of the high-throughput GridION X5 and PromethION devices make them suitable for any laboratory, while the high-performance compute modules allow real-time data analysis — even at full sequencing capacity — and reduces the potential burden of installing additional data analysis hardware.

In addition, Oxford Nanopore offers VolTRAX™, a compact, USB-powered automated library preparation device, which further simplifies the sequencing workflow (Figure 10).

The company is also developing the MinION Mk1C* — a fully integrated sequencing and analysis device (Figure 11). The MinION Mk1C combines the real-time, rapid, portable sequencing of MinION and Flongle with real-time, powerful computing and a high-resolution screen — offering a complete, go-anywhere solution for long-read DNA and RNA sequencing and analysis.

Figure 9MinIT: a simple, preconfigured IT solution for MinION and Flongle sequencing.

Figure 10VolTRAX: an automated library preparation solution for nanopore sequencing.

Figure 11MinION Mk1C: a fully integrated, fully portable sequencing and analysis device.*

* Available to pre-order. Anticipated availability Q3 2019.

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Case study 1Same-day genomic and epigenomic analysis of brain tumours

A plethora of technologies are currently required to assess different genomic and epigenomic alterations; however, the associated costs and long turnaround times combined with extensive infrastructure and training requirements have, to date, hindered their implementation5. To address these challenges, Dr. Philippe Euskirchen and co-workers at the ICM Brain and Spine Institute, France, assessed the potential of nanopore sequencing technology to deliver comprehensive and cost-effective characterisation of genetic alterations in brain cancer samples — including analysis of copy number (CN) alterations, epigenetic base modifications, and single nucleotide variations (SNVs)5,34. Furthermore, all nanopore sequencing workflows were designed to go from sample to result within a single day.

Using a low-pass whole genome sequencing approach, the team analysed both CN alterations and base modifications in a number of previously characterised brain tumour samples. The six-hour, real-time nanopore sequencing runs, which delivered <0.01x to 0.24x genome coverage, allowed detection of

chromosome arm-level CN alterations and epigenetic profiles with high concordance to matched microarray data (Figures 12 and 13). The low-pass methylation data allowed reliable subtyping of glioma samples into IDH-mutant and wild type. Mutations in the IDH genes typically result in global hypermethylation of CpG islands, which is associated with favourable prognostic outcomes. Significantly, the nanopore sequencing results were obtained within hours, as opposed to approximately 2 weeks as required for microarray-based analysis5.

Next, the team demonstrated the potential of the CN and methylation data to enable the identification of specific cancer types. All seven glioma samples tested were correctly identified using this approach. In addition, the team were able to determine the origin of several brain metastasis samples, including one instance of a breast adenocarcinoma metastasis found in the posterior fossa, for which immunohistochemistry had provided misleading results.

To detect known SNVs, the researchers designed an amplicon panel covering hotspot exons in IDH1, IDH2, and H3F3A, all coding exons of TP53 and, additionally, the TERT promoter (pTERT) region. Due to the long reads delivered by nanopore sequencing, this could be achieved with just nine PCR reactions. Using the real-time data acquisition capabilities of nanopore sequencing, the researchers were able to stop sequencing once the

‘Methylation data can directly be obtained from the same WGS data set which makes time-consuming bisulfite conversion and specialized methylation assays (sequencing or hybridization-based) expendable’5.

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low-pass whole genome sequencing (WGS) using a com-mercially available, handheld size nanopore sequencing device. With the aim of widespread implementation in routine diagnostics in mind, we used a transposon-based library preparation kit, which reduces sample preparation time to less than hour. In a cohort of 28 patients (Table 1), low-pass WGS for 6 h performed yielded a mean mapped read depth from <0.01X to 0.24X (Table S1), depending on the sequencing chemistry and input DNA fragment size. Nanopores decipher DNA sequence of single mol-ecules as they present to the pore, generating long reads of variable length, whose distribution is determined by DNA extraction and fragmentation method. We observed typical mean read lengths around 2 kb (Fig. 1b). As library preparation does not involve PCR amplification, no GC bias is introduced and the GC content distribution of the reads resembles closely that of the human refer-ence genome (Fig. 1c).

Copy number profiling

We then used WGS data to generate CN profiles. Reads were counted in 1000 kb windows, normalized and sub-jected to circular binary segmentation (Fig. 1c). No correc-tion of GC bias or mappability is necessary for nanopore reads; however, the long reads cause alignment artifacts with current reference genomes in regions with repetitive sequence such as centromeres. Still, the resulting CN pro-files closely resembled matched SNP array-based profiles (Fig. 1d). Importantly, codeletion of chromosome 1p/19q as a diagnostic criterion for oligodendrogliomas imple-mented in the 2016 WHO classification of CNS tumors was detected in three out of four affected samples (Fig. S1). The remaining sample did not yield sufficient read depth (<0.01) due to low input DNA quality (Table S1). High-level focal amplifications of EGFR, PDGFRA, and CDK4 were detected in affected glioblastoma samples (Table 1).

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Fig. 1 Copy number profiling using nanopore low-pass whole genome sequencing. a Same-day workflows to simultaneously char-acterize copy number variation (CNV) and methylation profiles or single nucleotide variants, respectively. Tumor DNA is subjected to quality control (QC), and then, 250 ng input material is used for library preparation for either whole genome sequencing (WGS) or PCR-based deep amplicon sequencing. b Representative read length distribution of mapped reads. Note log scale on X axis. c Representa-

tive distribution of GC content of reads in comparison with the hg19 human reference genome. A randomly drawn subsample of the entire reference genome split into 1000 bp fragments is shown. d Copy number profile showing log2 transformed, normalized read counts per 1000 kbp window (grey) with running mean (red) and segmentation results (blue). e Comparison of nanopore WGS with matched SNP arrays. Heatmaps indicate copy number calls (losses and deletions in blue, and gains and amplifications in red) across the genome

Figure 12Comparison of copy number profiles obtained using low-pass whole genome nanopore sequencing and SNP arrays for matched samples. Image adapted from Euskirchen et al 5 and available under Creative Commons license (creativecommons.org/licenses/by/4.0).

Figure 13Comparison of methylation calls from nanopore sequencing with matched Illumina 450K microarray data. Beta value distributions for CpG sites that were identified as unmethylated (red) or methylated (blue), respectively, by nanopore WGS are shown. Image adapted from Euskirchen et al 5 and available under Creative Commons license (creativecommons.org/licenses/by/4.0).

697Acta Neuropathol (2017) 134:691–703

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2, 4, 5, 12, 23, 24, 37–40]. Where CN data were available, too, SNP array-based CN profiles were aggregated to chro-mosome arm level and added to the training set (Fig. 3a). The resulting classifiers for any set of CpG sites in our

cohort usually yielded an overall out-of-bag classification error rate ≪5%.

We first subjected seven glioma samples with CN and methylation profiles generated by nanopore sequencing to

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Fig. 2 Methylome profiling by nanopore sequencing of native tumor DNA. a Comparison of methylation calls from nanopore sequenc-ing with matched Illumina 450K microarray-based data. Beta value distributions for CpG sites that were identified as unmethylated (red) or methylated (blue), respectively, by nanopore WGS are shown. b “Random taiga” simulation of classification error as a function of the number of randomly sampled CpG sites. Each dot represents the class-specific error rate of an ad hoc generated random forest using a

random subset of N CpG sites (indicated on X axis) from the TCGA lower grade glioma Illumina 450K cohort as training set. Lines indi-cate the mean of five independent simulations. c Methylation profiles from nanopore sequencing discriminate IDH-mutant and wild-type tumors. Bar plots indicate vote distribution from ad hoc random for-est classification. The TCGA low-grade glioma cohort was used as a training set. Illumina 450K-based beta values were dichotomized using >0.6 as threshold

desired read depth of 1,000x was reached for all amplicons, which was achieved in a timeframe of just 2–20 minutes. In all samples, coding mutations were reliably detected as compared to routine analysis based on Sanger sequencing, immunohistochemistry, or a next-generation sequencing (NGS) panel.

Furthermore, the facility for sample multi-plexing (WGS – 4 samples; amplicon – 12 samples) provided highly cost-effective analyses.

Confirming the advantaged of nanopore sequencing for cancer research, the team commented, ‘Nanopore sequencing allows same-day detection of structural variants, point mutations, and methylation profiling using a single device with negligible capital cost’ 5.

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Case study 2

Comprehensive and routine analysis of leukaemia samples

Compared to current methods for detecting gene fusions, such as real-time PCR and fluorescence in situ hybridisation (FISH), which take days or even weeks to perform, nanopore technology offers the advantages of rapid sequencing with real-time data generation.

With the aim of rapidly detecting and characterising a variety of oncogenic fusion events, including the BCR-ABL1 fusion (which is present in nearly all patients with chronic myeloid leukaemia [CML]), Jeck et al. developed a

workflow based on a modified Anchored Multiplex PCR (AMP) method for library construction. Initial proof of concept experiments in the K562 cell line using gene-specific primers against BCR exons 1 and 2, along with sequencing on the MinION, demonstrated the ability of this technique to precisely delineate the BCR-ABL1 fusion junction (Figure 14)7. Furthermore, real-time analysis enabled confident detection of the fusion within just 5 minutes, with the first fusion read being generated within five seconds.

Figure 14Method of library construction for MinION sequencing using Anchored Multiplex PCR (AMP). Adapted from Jeck et al 7.

1 │ Nanopore Community Meeting 2018 │ @NanoporeConf #NanoporeConf

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Based on these promising results, Jeck et al. applied an expanded AMP-based assay, targeting an array of oncogenic fusions, to a number of haematological malignancy specimens. Various fusion events were detected including PML-RARA — the hallmark of acute promyelocytic leukaemia. Sensitivity to this critical fusion was 100% in clinical research samples, even with a 1:10 dilution specimen. When multiplexing four samples on a fresh flow cell, all fusions could be detected within 6 hours of sequencing.

Application of this assay to previously characterised libraries from formalin-fixed, paraffin-embedded (FFPE) sarcoma specimens detected a range of gene fusions with high specificity, demonstrating that the problem of fragmentation and lower DNA quality in FFPE specimens did not impede accurate fusion detection using nanopore sequencing. Furthermore, the fraction of MinION reads mapping to a given fusion was higher than that observed with traditional short-read sequencing technology in all but one case, which the authors suggest is likely due to longer read length. The authors concluded that ‘…nanopore sequencing has great promise as a broad fusion detection platform…’7.

In a slightly different approach, Cumbo et al. compared FISH followed by Sanger sequencing to long-range template multiplex PCR and nanopore sequencing, to analyse BCR-ABL1 DNA fusions36. With a sequencing depth of 400x over the BCR region, the team observed concordance between the nanopore and Sanger sequencing results in all CML samples studied, stating that ‘…the very low costs, the ease of use, and the length of the reads (hundreds of kilobases), make MinION an ideal tool for target sequencing’ 36.

Researchers from the Fred Hutchinson Cancer Research Centre in Seattle, aimed to develop a single method allowing the detection and analysis of mutations in FMS-like tyrosine kinase 3 (FLT3), a tyrosine kinase receptor involved in haematopoietic cell proliferation, differentiation, and apoptosis33. Duplications in FLT3 are associated with aggressive acute myeloid leukaemia (AML). The quick workflows and real-time data acquisition afforded by nanopore sequencing offers a distinct advantage over traditional sequencing technologies. The researchers designed an RNA amplicon sequencing assay producing a 2,400 bp product covering well-defined hotspot regions in FLT3. MinION sequencing provided rapid acquisition of full-length reads, which allowed the reliable detection of internal tandem duplication mutations33.

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Case study 3

Characterising tandem repeats in dementia

Tandem repeats (TRs) have been implicated in a number of diseases, including Huntington’s, frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Such repeats can cause disease through their length, sequence or base modifications; however, these features are often difficult, if not impossible, to assess with traditional analysis technologies37. In addition, existing techniques only target a single tandem repeat locus at a time and have long turnaround times. Direct, long-read nanopore sequencing is capable of spanning large tandem repeats, revealing their size, nucleotide composition and the presence of base modifications. Furthermore, with the high-throughput, high-yield PromethION platform, this can be achieved at scale — allowing complete analysis of tandem repeats across multiple genomes.

To assess the facility of nanopore sequencing to fully resolve tandem repeats, researchers at VIB - University of Antwerp performed whole genome sequencing of 11 samples using the PromethION37,38. Uniformly high yields were obtained for all samples, with a maximum yield per flow cell of 98 Gb* (30x genome coverage). The team focused on the analysis of a variable number tandem repeat (VNTR) within the ABCA7 gene which they had recently discovered results in a >4-fold increased risk of Alzheimer’s disease.

All ABCA7 VNTR alleles, including those containing up to 10,000 bp expansions, were spanned by the long sequencing reads; however, existing nucleotide sequence aligners struggled to accurately resolve the length of these long tandem repeats. To circumvent these challenges the team developed NanoSatellite39, a novel algorithm that utilises raw nanopore “squiggle” data rather than basecalled sequence to characterise the repeats. This method allowed the detection of all clinically-relevant repeat expansions and was shown to deliver read length estimations commensurate with Southern blotting — the current ‘gold-standard’ analysis technique (Figure 15). Importantly, NanoSatellite also allowed the identification of two VNTR alleles for two individuals that appeared to be homozygous when using Southern blotting, emphasising the increased sensitivity of this technique.

Highlighting the benefits of the PromethION platform, the researchers commented: ‘Many tandem repeats in the human genome — some of which are currently uncharacterized — can be studied at once with a single sequencing run and somatic differences of unstable (expanded) TRs could be evaluated, which eventually will lead to the identification of novel disease-associated TRs and improved diagnostics’ 37.

* Flow cell performance continues to increase with current internal PromethION Flow Cell yields at Oxford Nanopore of 200 Gb (October 2018).

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Figure 15 Tandem repeat lengths called from raw nanopore signal data using NanoSatellite or nucleotide sequence using tandem-genotypes repeat caller (basecalled using either Albacore, Scrappie events, and Scrappie raw) were compared. In all cases, NanoSatellite delivered higher concordance with Southern blotting (dotted line). Positive strand (red); negative strand (blue). TG = tandem-genotypes repeat caller. Image adapted from De Roeck et al.37

The use of raw nanopore signal to characterise short tandem repeats has also been demonstrated by Gießelmann et al40. Employing a CRISPR-Cas12a enrichment approach, the team selectively targeted repeats within the C9orf72 gene (causative of frontotemporal dementia [FTD] and amyotrophic lateral sclerosis [ALS]) and the FMR1 gene (implicated

in fragile X syndrome). The in-house developed nanoSTRique analysis tool allowed precise characterisation of repeat length, while the amplification-free enrichment strategy further enabled analysis of epigenetic modifications — revealing significantly increased methylation at gene promoter CpG islands for the expanded alleles of both genes.

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Case study 4

Resolving a complex structural variant

Recent research has demonstrated that complex structural variations (cxSVs) — which involve three or more breakpoint junctions — are considerably more abundant and diverse than previously appreciated, with an average of 14 cxSVs detected in developmental disorder samples41. The advent of high-throughput genomic technologies has made the identification of cxSVs possible; however, detailed breakpoint analysis is still challenging, even for modern sequencing and microarray platforms.

At the University of Cambridge, UK, Alba Sanchis-Juan and colleagues are studying the association of cxSVs with Mendelian disease42. Using short-read sequencing technology they screened 1,342 samples from individuals with neurodevelopmental or retinal disorders to identify cxSVs that overlapped with clinically-important, disease-associated genes. Of the four samples identified, the nature of the variant could be resolved in all but one sample using a combination of short-read sequencing, Sanger sequencing and microarray analysis. Data for the unresolved sample, which came from a female neonate who presented with foetal bradycardia (low heart rate), suggested two alternative models of cxSV structure, in which either one or two disrupted copies of the gene CDKL5, associated with early infantile epileptic encephalopathy, were present (Figure 16).

To characterise the true nature and likely pathogenicity of the cxSV, the team turned to the long read sequencing capabilities offered by nanopore technology.

Using a low-pass whole genome sequencing approach on the MinION, a minimum of 4-fold coverage was achieved for all breakpoints, with an average read length of 8 kb. The long reads allowed phasing of the aberrant locus to be determined, identifying paternal inheritance. The phased reads also established the presence of an intact and disrupted copy of the CDKL5 gene, providing evidence to support the proposed model 1 (Figure 16). Furthermore, nanopore sequencing confirmed all the novel breakpoint junctions that could not be elucidated using Sanger sequencing due to the repetitive nature of the DNA, which precluded the design of specific primers. Subsequent gene expression analysis demonstrated that both CDKL5 alleles are expressed in the sample, indicating that the variant is likely benign.

‘Long-read whole genome sequencing is the best technology to look at breakpoint analysis in repetitive regions’ 43.

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*

A B C D E

A B C D EB D

A B EDBD C

A B C D E

A B C D E

A B ED C

B

B

D

D

280Kb 280Kb

Model 1 Model 2

Reference

Possibleintermediate

Derivative

458Kb

280Kb 280Kb

458Kb

A B C D E

A B C D EB D

A B EDBD C

Model 1

ONTreads(paternalallele)

Figure 16 Accurate resolution of a cxSV in a neonate sample using nanopore sequencing. Short-read sequencing technology detected a duplication-inversion-duplication event that overlapped with CDKL5 but was unable to resolve between two models of cxSV structure and formation. Long nanopore sequencing reads allowed accurate phasing and breakpoint resolution – confirming the existence of one intact and one disrupted copy of CDKL5 and supporting the proposed Model 1. Blue = duplications; green = inversions. Image adapted from Sanchis-Juan et al.43

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Case study 5

Rapid analysis of lower respiratory infection samples

Responsible for over three million deaths worldwide per year, lower respiratory infections (LRIs) are the leading cause of death from infectious disease44. A wide range of pathogens cause these infections but precise identification and characterisation of these organisms using current methodology can prove challenging.

The current ‘gold-standard’ approach for investigation of bacterial LRIs is culture, the results of which can be slow to obtain (48-72 hours) or uncertain30. Alternative PCR-based analysis methods reduce time to result but do not detect the whole spectrum of pathogens potentially present in a sample or their antimicrobial resistance (AMR) profiles. To address these challenges, researchers at the Quadram Institute Bioscience (QIB) in Norwich, and colleagues, assessed the utility of nanopore sequencing to provide rapid pathogen identification and antimicrobial resistance profiling from mixed, metagenomic samples30. According to the researchers: ‘Metagenomic sequencing-based approaches, which make no presumptions about the organisms and resistance genes that may be present, have the potential to overcome the shortcomings of both culture and PCR, by combining speed with comprehensiveness’ 30.

Key challenges of metagenomic sequencing include the presence of high levels of host DNA and lengthy time to result. To overcome these issues, the team developed a novel sequencing workflow that takes advantage of saponin-based host DNA depletion and the real-time data analysis afforded by nanopore sequencing. Using this workflow, up to 99.99% of host nucleic acids were removed from the samples, while pathogen detection was 96.6% concordant with traditional culture techniques (Table 2; Figure 17). Importantly, the entire process — from sample acquisition to pathogen and antibiotic resistance gene identification — was achieved within just six hours. Furthermore, continuation of the sequencing run allowed the generation of sufficient data for reference-based pathogen genome assemblies, which the researchers suggested would enable investigations into emergence and spread of pathogens30.

This novel nanopore sequencing workflow is currently being evaluated as part of the INHALE programme, which aims to assess the potential of several molecular techniques to rapidly characterise the organisms responsible for pneumonia45.

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PathogenCulture positive

Metagenomic

DetectedNot

detectedAdditional detection

Haemophilus influenzae 10 10 0 4

Staphylococcus aureus 8 8 0 1

Pseudomonas aeruginosa 6 5 1 1

Escherichia coli 3 3 0 1

Moraxella catarrhalis 2 2 0 3

Serratia marcescens 2 2 0 0

Klebsiella oxytoca 1 1 0 0

Klebsiella pneumoniae 1 1 0 1

Streptococcus pneumoniae 0 0 0 6

None (NRF/NSG) 0 0 0 10

Table 2High concordance was demonstrated between nanopore metagenomic sequencing and routine culture positive results. The metagenomic approach also allowed the identification of additional bacteria not reported using culture. Table adapted from Charalampous et al 30.

Figure 17Real-time species identification and quantification was performed using the WIMP workflow from Oxford Nanopore, which enabled easy visualisation of results. Images courtesy of Dr. Justin O’Grady, University of East Anglia, UK.

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OXFORD NANOPORE TECHNOLOGIES | THE PROMISE OF NANOPORE SEQUENCING FOR CLINICAL RESEARCH

A complementary approach to rapidly characterise antibiotic resistance in metagenomic LRI and other clinical research samples was recently demonstrated by Břinda et al46. This study presents a novel method for inferring antibiotic resistance based on the identification of DNA sequence variation

detected in a previously characterised antibiotic-resistant bacteria. Using nanopore sequencing, the team was able to sequence all bacteria present in a sample in sufficient depth to identify a known antibiotic resistant strain within just 5 minutes.

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Case study 6

Metagenomic analysis of viral outbreaks

In recent decades, human disease outbreaks caused by arboviruses have increased in prevalence. Arboviruses are predominantly RNA viruses that replicate within blood-sucking arthropod vectors. Dengue virus (DENV) and chikungunya virus (CHIKV), transmitted to humans via Aedes species mosquitoes, are two arboviruses of increasing concern as they no longer require enzootic amplification and have been responsible for widespread epidemics47.

DENV and CHIKV are single-stranded positive-sense RNA viruses with overlapping geographical distributions. Differential identification based on symptoms is difficult as the clinical presentations are similar (e.g. high fever, rash, headache, myalgia)47. Therefore, rapid and unbiased identification methods are vital for determining the pathogen responsible for an outbreak, for real-time genomic surveillance and the ability to discriminate co-infection.

Metagenomic sequencing using nanopore technology has the power to detect and monitor syndromic outbreaks in a single unbiased and real-time assay. Kafetzopoulou and colleagues tested the feasibility and sensitivity of direct metagenomic sequencing of DENV and CHIKV genomes in serum and plasma samples infected with a range of viral loads47,48. Four samples for each virus were selected for nanopore sequencing, which provided a 99% genome coverage at a depth of 20x, even for samples

with very low viral titres (DENV 31.29Ct; CHIKV 32.52Ct). High concordance was observed between short-read and nanopore sequencing data (Figure 18). Importantly, using nanopore sequencing, near maximum coverage for both viruses was obtained within just 8 minutes when using the 1D2 Sequencing Kit or within 85 minutes when using the Rapid Sequencing Kit for library preparation. Furthermore, in one CHIKV sample the researchers identified the presence of DENV, highlighting the significant advantage that nanopore sequencing has over targeted techniques in its ability to detect co-infection. Taken together, these results demonstrate the sensitivity and speed that nanopore sequencing technology provides, and the researchers conclude that nanopore technology is ‘…ideally suited for the investigation of viral species with high levels of genetic diversity which are difficult to assess using targeted methods’ 47.

A significant advantage of nanopore sequencing using the portable MinION device is the facility for direct field application, which allows rapid deployment and negates the need to ship samples back to the laboratory. For example, in-field genomic surveillance of an ongoing Ebola epidemic was achieved by Quick et al. through successful application of nanopore sequencing technology27. Kafetzopoulou and colleagues travelled to the Institute of Lassa Fever Research and Control in Nigeria, a remote and resource-

23


Figure 18 Comparison of the proportion of viral reads and reference genome coverage between nanopore and short-read sequencing data, demonstrating highly concordant results between the techniques and near 100% coverage at low viral titres. For each sample, identified by Ct value, the percentage of total reads mapping to the appropriate reference sequence, and therefore attributed as viral in origin, is plotted in the upper panels. Lower panels display the percentage of the reference genome sequenced to a minimum depth of 20-fold in the data generated. Adapted from Kafetzopoulou et al 47.

limited location, to apply their nanopore sequencing workflow to in-field monitoring of a Lassa viral outbreak49. Lassa fever is endemic in Nigeria and the virus is transmitted via contact with infected rats. The Lassa virus genome is highly divergent and current detection requires two real-time, reverse transcription PCRs to cover all the possible variants of Lassa being circulated. Unbiased, rapid metagenomic analysis using nanopore technology is ideally suited for such situations, where investigation of highly genetically diverse viral species is cumbersome using targeted methods. During their time in Nigeria, the team encountered the largest Lassa fever outbreak ever reported. They established

a nanopore sequencing and analysis workflow in a resource-limited setting, delivering complete genomic analysis of 35 samples within 21 days from the request of sequencing information on circulating strains by the Nigerian Centre of Disease Control. Phylogenetic analysis of the virus indicated that the outbreak was due to independent spill-over from the rodent reservoir, with no evidence of extensive human-to-human transmission. Kafetzopoulou and colleagues concluded that their workflow ‘…confirms the feasibility of field metagenomic sequencing for these and likely other RNA viruses, highlighting the applicability of this approach to front-line public health’ 49.

short-read MinION

short-read MinION

24


Summary

Sequencing holds great promise for the identification of novel diagnostic and prognostic disease markers that can inform the next generation of clinical assays; however, the nature of traditional short-read sequencing technologies makes it exceptionally challenging to analyse many important classes of disease-associated genomic variants, including large SVs, repeats, phasing, fusion transcripts, and base modifications.Long-read, real-time nanopore sequencing overcomes these challenges, providing rapid analysis of a broader range of genomic variants.

Offering cost-effective portable and high-throughput benchtop devices, nanopore technology can be accessed by any researcher in any environment, accelerating advances in human health.

3

25


Oxford Nanopore Technologies introduced the world’s first nanopore DNA sequencer, the MinION — a portable, real-time, long-read, low-cost device — followed by the larger GridION X5, PromethION P24 and P48 devices, and smaller Flongle (Table 3). The long, direct reads offered by nanopore sequencing deliver more comprehensive analysis of human genetic variation,

About Oxford Nanopore Technologies4

allowing enhanced characterisation of SV, repetitive regions, haplotype phasing, RNA splice variants, isoforms and fusion transcripts. These benefits can be applied across whole genome, whole transcriptome and targeted sequencing approaches, allowing researchers complete flexibility in the development of cost-effective genomic analysis workflows.

Flongle MinION GridION X5PromethION (1 flow cell)

PromethION P24/P48 (24 or 48 flow cells)

Read length Fragment length = read length. Longest read now >2 Mb

Run time 1 min - 24 hrs 1 min - 72 hrs 1 min - 72 hrs 1 min - 64 hrs 1 min - 64 hrs

Theoretical maximum 1D Yield

Up to 3.3 Gb Up to 50 Gb Up to 250 Gb Up to 315 Gb Up to 7.5 Tb / Up to 15 Tb

Current yield range (customer best January 2019)

Up to 1.8 Gb Up to 30 Gb Up to 150 Gb Up to 160 GbUp to 3.8 Tb / Up to 7.6 Tb

Multiplexing enabled Not in first release

Kits for 96 samples

Kits for 96 samples

Kits for 24 samples

Kits for 24 samples

Number of channels available for sequencing

Up to 126 Up to 512 Up to 2,560 Up to 3,000 Up to 72,000 /Up to 144,000

Table 3 Oxford Nanopore devices combine high yields with a range of throughputs to suit all sequencing requirements.

For the latest information about the use of nanopore technology in clinical research, visit www.nanoporetech.com/applications.

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51. Payne, A., Holmes, N., Rakyan, V. and Loose, M.

Whale watching with BulkVis: A graphical viewer for Oxford Nanopore bulk fast5 fles. bioRxiv 312256 (2018).

2. De Roeck, A. Human genome sequencing on PromethION to investigate tandem repeats in dementia. Presentation. Available at: https://nanoporetech.com/resource-centre/human-genome-sequencing-promethion-investigate-tandem-repeats-dementia [Accessed: 07 February 2019]

3. Brandler, W.M. et al. Paternally inherited cis-regulatory structural variants are associated with autism. Science. 360(6386):327-331 (2018).

4. Ishiura, H. Expansions of intronic TTTCA and TTTTA repeats in benign adult familial myoclonic epilepsy. Nat Genet. 50(4):581-590 (2018).

5. Euskirchen, P. et al. Same-day genomic and epigenomic diagnosis of brain tumors using real-time nanopore sequencing. Acta Neuropathol. 134(5):691-703 (2017).

6. Gong, L. et al. Picky comprehensively detects high-resolution structural variants in nanopore long reads. Nat Methods 15(6):455-460 (2018).

7. Jeck, W.R. et al. A nanopore sequencing–based assay for rapid detection of gene fusions. J Mol Diagn. S1525-1578(17)30630-X (2018).

8. Cretu Stancu, M. et al. Mapping and phasing of structural variation in patient genomes using nanopore sequencing. Nat Commun. 8(1):1326 (2017).

9. Leija‐Salazar, M. et al. Evaluation of the detection of GBA missense mutations and other variants using the Oxford Nanopore MinION. Mol Genet Genomic Med. e564 (2019).

10. Taylor, G. Nanopore sequencing in clinical diagnostics. Presentation. Available at: https://nanoporetech.com/resource-centre/nanopore-sequencing-clinical-diagnostics [Accessed: 09 February 2019]

11. Jain, M. et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol. 36(4):338-345 (2018)

12. Liu, C. et al. Accurate typing of Human Leukocyte Antigen Class I genes by Oxford Nanopore sequencing. J Mol Diagn. 20(4):428-435 (2018).

13. Nieto, T. How nanopore sequencing is changing HLA typing for renal transplants in low income countries. Presentation. Available at: https://nanoporetech.com/resource-centre/tom-nieto-how-nanopore-sequencing-changing-hla-typing-renal-transplants-low-income [Accessed: 09 February 2019]

14. Orsini, P. et al. Design and MinION testing of a nanopore targeted gene sequencing panel for chronic lymphocytic leukemia. Sci Rep. 8(1):11798 (2018)

15. Steijger, T. et al. Assessment of transcript reconstruction methods for RNA-seq. Nature Methods 10, 1177–1184 (2013)

16. Clark, M. et al. Long-read sequencing reveals the splicing profle of the calcium channel gene CACNA1C in human brain. bioRxiv 260562 (2018).

17. Hardwick, S.A. et al. Targeted, high-resolution RNA sequencing of non-coding genomic regions associated with neuropsychiatric functions. bioRxiv 539882 (2019).

18. Singh, M. et al. High-throughput targeted long-read single cell sequencing reveals the clonal and transcriptional landscape of lymphocytes. bioRxiv 424945 (2018).

19. Keller, M.W. et al. Direct RNA sequencing of the coding complete influenza A virus genome. Sci Rep. 8(1):14408 (2018)

20. Weng, Y.L., An, R., Shin, J., Song, H., and Ming, G.L. DNA modifications and neurological disorders. Neurotherapeutics. (4):556-67 (2013)

21. Simpson, J.T. et al. Detecting DNA cytosine methylation using nanopore sequencing. Nature Methods 14: 407–410 doi:10.1038/nmeth.4184 (2017).

22. Smith, A.M. et al. Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing. bioRxiv 132274 (2017).

23. Gilpatrick, T. Cas9 targeted enrichment for nanopore profiling of methylation at known cancer drivers. Presentation. Available at: https://nanoporetech.com/resource-centre/cas9-targeted-enrichment-nanopore-profiling-methylation-known-cancer-drivers [Accessed 11 February 2019]

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26. Liu, H. et al. Accurate detection of m6A RNA modifications in native RNA sequences. bioRxiv 525741 (2018).

27. Quick, J. et al. Real-time, portable genome sequencing for Ebola surveillance.Nature 530(7589):228-232 (2016).

28. Votintseva, A. A. et al. Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples. J Clin Microbiol 55(5):1285-1298 (2017).

29. Rhodes, J. et al. Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen Candida auris. Emerg. Microbes Infect. 7(1):43 (2018).

30. Charalampous, T. et al. Rapid diagnosis of lower respiratory infection using nanopore-based clinical metagenomics. bioRxiv 387548 (2018).

31. Kafetzopoulou, L.E. et al. Assessment of Metagenomic MinION and Illumina sequencing as an approach for the recovery of whole genome sequences of chikungunya and dengue viruses directly from clinical samples. Euro Surveill. 23(50) (2018).

32. Jeck, W.R. Nanopore sequencing and rapid fusion testing – a ‘killer app’ in molecular pathology. Presentation. Available at: https://nanoporetech.com/resource-centre/william-jeck-nanopore-sequencing-and-rapid-fusion-testing-killer-app-molecular [Accessed: 07 February 2019]

33. Yeung, C. and Sala-Torra, O. Clinical applications for real-time sequencing in leukaemia. Presentation. Available at: https://nanoporetech.com/resource-centre/direct-rna-cdna-sequencing-human-transcriptome. [Accessed: 07 February 2019]

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43. Sanchis-Juan, A. Complex structural variants resolved by short-read and long-read whole genome sequencing in Mendelian disorders. Presentation. Available at: https://nanoporetech.com/resource-centre/complex-structural-variants-resolved-short-read-and-long-read-whole-genome-0 [Accessed: 30 October 2018]

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47. Kafetzopoulou, L.E. et al. Assessment of Metagenomic MinION and Illumina sequencing as an approach for the recovery of whole genome sequences of chikungunya and dengue viruses directly from clinical samples. Euro Surveill. 23(50) (2018).

48. Kafetzopoulou, L.E. et al. Metagenomic sequencing at the epicenter of the Nigeria 2018 Lassa fever outbreak. Science. 363(6422):74-77. (2019).

49. Kafetzopoulou, L.E. Metagenomic nanopore sequencing: RNA viruses from lab to field. Presentation. Available at: https://nanoporetech.com/resource-centre/metagenomic-nanopore-sequencing-rna-viruses-lab-field [Accessed: 30 October 2018]

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Oxford Nanopore Technologies, the Wheel icon, GridION, Flongle, Metrichor, MinION, MinIT, MinKNOW, PromethION, SmidgION and VolTRAX are registered trademarks of Oxford Nanopore Technologies in various countries. All other brands and names contained are the property of their respective owners. © 2019 Oxford Nanopore Technologies. All rights reserved. Oxford Nanopore Technologies products are currently for research use only.

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