Eukaryotic genomes are organized non-randomly. We are studying how abrupt changes to genome organization shapes the transcriptional landscape using Sc2.0, a yeast strain composed entirely of designer, synthetic DNA. Synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) is a key design feature of the synthetic yeast genome project. SCRaMbLE generates stochastic and complex rearrangements at engineered recombinase sites throughout the genome on-demand using Cre-mediated recombination. We have characterized extensive transcriptional diversity in >60 independent SCRaMbLE strains containing a circular synthetic chromosome, synIXR. These transcriptional alterations appear to be isolated to the SCRaMbLEd chromosome and dependent on rearrangements specific to each SCRaMbLE strain. Using Oxford Nanopore’s direct RNA sequencing, we have observed novel transcriptional events in SCRaMbLEd genomes, including alteration to transcript start and termination sites. Our results suggest an inextricable link between physical organization of the genome and transcript isoform expression.  

Nanopore sequencing technology can be applied to identify the pathogen responsible for an outbreak through sequencing all nucleic acids existing in the collected sample in a single run. In addition, it gives insight about the origin and variant of the causative agent. We have established a novel sequencing protocol relying on nanopore sequencing and offline BLAST search beside a microbiome screening of an ancient tomb. The whole procedure was conducted in a solar powered mobile suitcase laboratory, which is easy to use at the point of need. The procedure was completed in 5 hours including extraction, barcoding, sequencing and data analysis, which did not require a bioinformatician. Our protocol enables rapid and reliable foot and mouth disease virus serotyping and the differentiation of the Capri poxviruses (Sheep poxvirus, Goat poxvirus and Lumpy Skin Disease virus). The microbiome composition of the ancient tomb revealed potential threat of respiratory illness due to bacteria from family of Bacillaceae. Furthermore, bacteria from family of Pseudomonadaceae gave hints to the former use of the tomb as a byre.

Ruminants such as cows and sheep are important livestock species. They convert low nutritional value plant matter into high-quality meat and dairy products. Within a specialised stomach called the rumen, microbes ferment the plant matter producing short-chain fatty acids from difficult to digest plant matter. The composition of the rumen microbial community can affect the animal’s health, feed efficiency and level of methane production. Species in the rumen are typically difficult to culture and despite its importance, it remains an underexplored environment. DNA sequencing of the contents of the rumen offers the potential to identify microbial species without culture techniques. Here we sequence cow rumen fluid using Oxford Nanopore sequencing. We show that despite these data coming from a highly complex microbial sample we can assemble high-quality, single-contig whole genomes and plasmids of known and novel species, including numerous circular contigs. Additionally, we compare and validate the assemblies of these genomes with binned genomes generated from short read Illumina assemblies. We show that the long-read assembly out performs the short-read assembly in contiguity and in incorporation of important features such as AMR genes and marker genes..

Acute myeloid leukemia (AML) represents clonal expansion of malignant cells. A stratification of patients in risk groups is based on cytogenetics and molecular markers for a genotype-based treatment strategy. Conventional karyotyping, which is necessary for classification of “high-risk” AML, is available after 5 to 7 days. Using Oxford Nanopore sequencing, we established karyotyping based on shallow genome sequencing within 24 hours. The throughput of one flowcell was sufficient to achieve 3-fold genome coverage and reproduce results of conventional karyotyping in 20 AML patients. To discover structural variations, we applied direct RNA sequencing and analysed fusion genes based on 1.2 million reads. A single run is sufficient to detect a balanced translocation t(9;22), a fusion gene BCR-ABL1, in the cell line K-562. While a study of a larger AML patient cohort is ongoing, parallel low coverage genome and transcriptome analysis allows identification of high-risk AML during the initial diagnostic work-up of 24 hours.

It is well-known that patients diagnosed with chromosomal structural abnormality will lead to increased miscarriage rate. Preimplantation genetic testing for chromosomal structural rearrangement (PGT-SR) can increase the pregnancy rate, and provides a chance to avoid the genetic defect in the successive generation if handles appropriately. Diagnosis of the DNA breakpoint is always a difficult task from both effective and economical point of views. In the past, we performed haplotype analysis to phase the disease and wild-type chromosomes. Although it is feasible, the occurrence of double crossover between breakpoints and markers can generate false negative result. In this session, I will demonstrate the approach of breakpoints determination using latest technology advancement that maximize the efficiency and minimize the economic burden. Precise identification of breakpoints simplifies PGT-SR to an approach similar to PGT-M (preimplantation genetic testing – Monogenic disease). This strategy requires only a couple of PCR reactions to distinguish the derivative from wild-type chromosomes accurately. Moreover, such breakpoint information is applicable to all family members affected by the same chromosomal aberrations, where haplotype approach require reassessment of microsatellite markers for every couple even shared the same chromosome aberrations.

Viral genomes typically exhibit a higher gene density and more diversified transcriptome than the host cell, despite their smaller, more constrained size. Coding potential is maximized by the use of overlapping open reading frames, alternative polyadenylation, and/or the deployment of complex splicing patterns. These properties make it more challenging to accurately dissect viral transcriptomes and there is a need for new tools and approaches that either complement or supplant conventional approaches. Here, we demonstrate how direct RNA sequencing of polyadenylated RNAs collected from primary cells infected with herpesviruses enables us to examine splicing patterns, transcription initiation and termination, RNA editing, and the placement of RNA modifications for both host and viral transcripts – all performed without inherent bias and at the single molecule level. We examine the pros and cons of using Illumina sequencing to perform error-correction and how ‘pseudo-transcripts’ can be used to examine the protein coding potential of individual RNAs. In addition to cataloguing novel splice variants of canonical transcripts, we have also discovered a number of novel non-coding RNAs that regulate viral infections. Most surprisingly, we have uncovered a novel class of transcript that results from read-through transcription and back-splicing to produce viral fusion proteins. Taken together, direct RNA sequencing offers an invaluable tool for dissecting complex viral transcriptomes leading to new biological understanding. 

Short-read sequencing platforms have been adopted by public health agencies for infectious disease surveillance worldwide and have proved to be a robust and accurate method for quantifying relatedness between bacterial genomes. However, this approach offers less flexibility for urgent, small scale sequencing that is often required during public health emergencies. In contrast, Oxford Nanopore Technologies offers a range of rapid real-time sequencing platforms, although at this time it has been suggested that lower read accuracy compared to other sequencing technologies might be problematic for variant identification. We compared Illumina and Oxford Nanopore sequencing data of two isolates of Shiga toxin producing Escherichia coli to assess the utility of nanopore technologies for urgent, small scale sequencing.  We investigated whether the same single nucleotide variants were identified by the two sequencing technologies and whether inference of relatedness was consistent.  We show that with optimised variant calling using nanopore sequencing data alone, it is possible to rapidly determine whether or not two cases of were likely to be epidemiologically linked.

The DNA within the nucleus of an interphase cell is organised into a complex hierarchy of folds and loops known as the 3D Genome. The development of various chromatin conformation capture methods has enabled the detection of the structures that define each level of this hierarchy e.g. chromosome territories, A/B compartments, topologically associated domains (TADs) and promoter-enhancer loops. This in turn has facilitated functional studies which have uncovered some of the mechanisms behind the formation and maintenance of these structures, as well as their effect on gene expression. However, most of these studies rely on methods that could only capture interactions between two points on the genome, and thus lacked the ability to resolve higher-order interactions. We will share our progress on Pore-C, a method to generate genome-wide, multi-contact chromatin conformation maps. We will also demonstrate how it can be used to improve whole genome assemblies and help resolve complex structural variants in cancer.

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that targets mRNAs with premature termination codons, as well as some endogenous mRNAs that encode full-length proteins. The features that render an mRNA sensitive to NMD are still poorly understood, except for the presence of an exon junction complex (EJC) >55 nts downstream of the termination codon. Obscuring the identification of NMD-inducing features is the fact that previous transcriptome-wide analyses of endogenous NMD targets did not reveal which specific splice isoforms are degraded by NMD. This is mostly attributed to the insufficient coverage of splice junction sites and the lack of information regarding non-annotated mRNA isoforms that are enriched upon NMD inhibition. A recent comparative transcriptome analysis from our lab of cells, in which three essential NMD factors were knocked down and then rescued, identified a high-confidence set of genes whose transcripts react to NMD (Colombo et al., RNA, 2016). However, because the analysis was based on short-reads only, we could not obtain reliable isoform-specific information. For an isoform-specific analysis, we now use cDNA nanopore sequencing, which allows us to identify full-length mRNAs that are stabilized upon NMD inhibition. Our approach can detect full-length isoforms that are enriched, or even appear, when NMD is inactivated and we have experimentally verified several examples. We integrate long and short-read sequencing to accurately quantify the expression of individual isoforms and thereby identify those that are targeted by NMD. We aspire to reveal the regulatory role of NMD at isoform-specific level and generate a resource that will enable the study of features that render a specific mRNA sensitive to NMD.

With thousands of fatalities due to car collisions with wildlife reported each year, roadkill are an underexploited resource in genomics. Here we show that mammalian roadkill samples could be used as a suitable source of DNA for long-read sequencing using the MiniON device for two carnivoran species frequently encountered along South African roads: the bat-eared fox (Otocyon megalotis) and the aardwolf (Proteles cristatus). For both species, hybrid assembly of 150PE Illumina reads at ~85X coverage (~215 Gb) and MiniON long reads at ~12X coverage (~30 Gb) using the MaSuRCA assembler provided genomes with high contiguity (~10,000 contigs with N50 of ~700 Kb) and completeness (>90% of complete BUSCOs). We further demonstrate that about 90% of the 14,509 single-copy orthologous genes of the OrthoMaM database could be successfully retrieved from these assemblies. These figures compare favourably with current mammalian genome assemblies and set our genomes among the best carnivore genomes currently available. This cost-effective strategy to obtain high quality reference mammalian genomes opens the way for large-scale population genomic studies of mammalian wildlife using resequencing of samples collected from roadkill. We illustrate the potential of the approach for genome scale species delimitation in both species for which subspecies have been defined based on disjunct distributions and morphological differences.

Human genes contain many long introns with degenerate sequence information at splice sites, requiring sophisticated mechanisms to locate and coordinate the excision of multiple introns within the same pre-mRNA transcript. Fundamental aspects of this process remain unexplored due to a lack of quantitative approaches that monitor RNA processing as transcripts are produced. Here we performed nanopore sequencing of nascent, or newly synthesized, RNA to directly probe the timing and patterns of mRNA splicing. Direct RNA sequencing by the Oxford Nanopore Technologies MinION reveals the native context of long RNA molecules from 3’ to 5’ without amplification-associated biases. By combining direct RNA nanopore sequencing with stringent purification of nascent RNAs, we measure both the active transcription site (nascent RNA 3’ ends) and the splice isoform of single RNA molecules as they are transcribed. Application to human K562 cells reveals that co-transcriptional splicing occurs after RNA Polymerase II has transcribed several kilobases past the 3’ splice site of most introns. We also observe that the order of intron removal is not influenced by transcription direction in human cells. By contrast, we analyzed nascent RNA from Drosophila S2 cells, which have a different gene structure, and found that co-transcriptional splicing occurs more rapidly and in the order of transcription. Treating cells with the splicing inhibitor Pladienolide B abolishes co-transcriptional splicing in both species. Altogether, directly sequencing nascent RNAs through nanopores exposes critical molecular processes that occur during transcription in living cells.

Despite reductions in malaria prevalence in the last two decades, the World Health Organization still reported an estimated 435 thousand deaths in 2017, the majority occurring in children under the age of five. Moreover, continued progress is threatened by emerging drug and insecticide resistance. Our team won the 2019 Land Rover Bursary, supported by the Royal Geographic Society, on a proposal to convert a 2019 Land Rover Discovery into a mobile sequencing lab and drive it 6300km across Africa, from the Atlantic to Indian Ocean. During our journey, we met with local research teams and policy makers striving to combat malaria, and produced materials aiming to raise public awareness and keep malaria on the global development agenda. My role in the project was to develop and pilot the mobile lab which, with local collaborators, we used to sequence antimalarial resistance genes in Zambia, and whole mosquito genomes in Kenya. We hope our project promotes the feasibility of a decentralized approach to pathogen and vector sequencing and marks the beginning of long-term collaborations incorporating in-country nanopore sequencing with policy-directed malaria research.

Assemblies of small eukaryotic genomes using long-reads are often close to complete. However, these assemblies remain difficult to validate, especially when genomes have complex features such as large inversions, translocations, ploidy variations, and where chromosome number may not be known. While many tools for assessing assemblies with short-reads exist, long-reads have far greater power for confirming the accuracy and completeness of contigs. I will present Tapestry, a tool for validating the contigs of a small assembly automatically and visualising the contigs so the structure of the assembly can be refined before polishing. I will show how Tapestry has helped us to resolve the complex genomes of several small eukaryotes.

At present, most metagenomic surveys are performed using short-read sequencing. This approach limits the specificity of taxonomic assignment and result in highly fragmented assemblies. Single molecule sequencing platforms are able to sequence much longer molecules and the output of these platforms, particular the PromethION from Oxford Nanopore, now supports the study of complex microbial communities using shotgun metagenomics. We assessed a variety of commercially available and manual extraction methods using both a ten-species mock community and clinical samples of stool to find a method capable of generating ultra-long reads (>100 kb). Neither bead-beating or column-based extraction methods were found to support reads of the desired length and moving to magnetic bead and manual extraction methods allowed significant improvements in read-length. We also demonstrated the power of using solely chemical and enzymatic cell lysis methods for extracting high-molecular weight DNA from recalcitrant organisms, such as Gram-positive bacteria and fungi, over popular physical disruption methods. Development of these methods is critical to support the growing field of clinical microbiome research, including the ability to perform strain tracking and produce high-quality metagenome assembled genomes (MAGs) from metagenomic samples.

Single cell transcriptome sequencing has become a powerful tool for high-resolution analysis of gene expression in individual cells. However, current high throughput approaches only allow sequencing of one extremity of the transcript (transcriptome profiling). Information crucial for an in-depth understanding of cell-to-cell heterogeneity on splicing, chimeric transcripts and sequence diversity (SNPs, RNA editing, imprinting) is lost. Here we present an approach that uses Oxford Nanopore sequencing with unique molecular identifiers to obtain error corrected full length single cell sequence information with the 10xGenomics single cell isolation system and apply it to examine differential RNA alternative splicing and RNA editing events in the embryonic mouse brain. 

Retrotransposons are transposable genetic sequences that copy themselves into an RNA intermediate and insert elsewhere in the genome by reverse transcription. Almost half of the human genome is derived from transposon derived sequences but only some dozens of full length Long Interspersed Nuclear Element-1s (LINE1s) in the human germline are expected to be retrotransposition competent. Mapping and genotyping retrotransposons with short-reads is complicated due to their size and high copy number of their consensus sequence in the reference genome, so we applied nanopore sequencing to study multiple aspects of LINE1 retrotransposition. First, we sequenced Inverse-PCR products with MinION detecting highly subclonal insertion sites of a particular LINE1 element in two colon cancer tumors. Second, we have whole genome sequenced few germline and tumor genomes with PromethION, detecting the whole range of retrotransposon insertions that are variable within humans or inserted during tumorigenesis. Finally, we studied DNA methylation around the LINE1 insertion and source sites in human tumors in order to understand the mechanisms of LINE1 activation in during tumorigenesis. Presently we are extending these studies to ~300 whole genomes of Uterine Leiomyoma tumors and their respective normal sequences.

GRAID is the Global Research Alliance for Infectious Disease, a collaborative international effort for infectious disease research using MinION. In this framework, we try to educate researchers and develop the methods and guidelines for field analysis of many aspects of infectious disease. We have conducted four summer schools in three developing countries: Thailand, Indonesia, and Kenya, as part of our efforts to introduce MinION in those countries. We are collaborating with researchers and have produced papers about serotype identification of dengue virus and comprehensive drug resistance identification of malaria parasites. Using MinION and isothermal amplification, we identified serotype of dengue virus in Manado, Indonesia and Hanoi, Vietnam. We found that this method simplifies the amplification of the virus nucleic acid by using only blood or serum and a water bath or a thermal block prior to library preparation. We analyzed 141 Indonesian and 80 Vietnamese patients. The overall successful detection rate was 79% and it depends largely on the viral titer. We also determined that the serotype of dengue virus is different in Indonesia and Vietnam, which is DENV1 and DENV3, respectively. Our next collaborative project is to comprehensively describe the drug resistance of malaria parasites in Indonesia, Vietnam, and Thailand. Here, we used PCR to amplify nine genes correlated to the drug resistance phenotype. We sequenced 118, 11, and 5 samples from Indonesia, Thailand, and Vietnam in multiplex manner and described the drug resistance pattern in each country. We found a position in K13 gene non-propeller region mutated quite frequently from our Indonesian samples. Although we believe that the mutation is not related to artemisinin resistance, we think that the parasites may be on selective pressure due to the artemisinin administration in the region. We also are working with bioinformaticians to develop a graphical user interface tools for researchers or clinicians who are unfamiliar with bioinformatics analysis. We have published Nano Pipe, which serves as an easy to use MinION data analysis tool for a ‘regular’ user. We have ongoing and prospective projects in the framework, such as HLA typing in the severe dengue patients, identification of unknown fever-causing pathogens, and determining the drug resistance pattern in HIV. We are confident that our consortium will make an impact in the infectious disease community to switch to sequencing in the research context while laying some foundations for preventive or therapeutic medicine in the future. 

We have harnessed nanopore sequencing to study DNA replication genome-wide at the single-molecule level. Using in vitro prepared DNA substrates, we characterized the effect of bromodeoxyuridine (BrdU) substitution for thymidine on the MinION nanopore electrical signal. Using a neural-network basecaller trained on yeast DNA containing various amount of BrdU, we identified BrdU-labelled tracts in yeast cells synchronously entering S phase in the presence of hydroxyurea and BrdU. As expected, the BrdU-labelled tracts coincided with previously identified early-firing, but not late-firing, replication origins. After BrdU pulse-labelling of asynchronous cells, we could detect and orientate dozens of thousands of individual replication tracts. This allowed us to reproduce RFD profiles obtained by OK-seq and to map thousands of initiation events, the vast majority of them coinciding with the well characterized ARSs. These results open the way to high-throughput, high-resolution, single-molecule analysis of DNA replication in many experimental systems.

River confluence and their microbial dynamics are least explored throughout globe. River Ganges is one of the most important and holy rivers of India and has a great mythological history and important for mass bathing events. Mainly Yamuna River is known to meet Ganges to form a confluence at Prayagraj (Allahabad), India. However, the influence of Yamuna river on taxonomic and functional profiling of microbial communities at the confluence of Ganga and Yamuna and in the succeeding downstream of confluence remain uncharacterized. Therefore, in year 2017 we undertook mega study under the directives of Government of India’s River cleaning mission (under the aegis of National Mission for Clean Ganga) and network program led by Council of Scientific and Industrial Research (CSIR) and Indian Council of Medical Research (ICMR) to understand India’s largest and sacred river i.e. Ganges. Water and sediment samples collected from Ganga-Yamuna confluence were processed for deciphering microbiome diversity and taxonomic functions using MinION sequencing technology. Preliminary investigations of confluence microbiome at Parayagraj revealed similar taxonomic (bacterial, fungal, archeal, and phages) and functional (resistomes) microbial profiles in the upstream (of Ganges) and farther downstream sites of the confluence revealed a transient influence of Yamuna River on holy River Ganges. Overall, Ganges River harbors plethora of microbial diversity and hidden treasure of functional potential that could be useful to depict non-putrefying properties of this river.

After seeds mature on the mother plant, they contain all the molecular machinery they will need for germination. Generally, some time elapses between maturation and germination. At the National Center for Genetic Resource Preservation, this time may be months to decades, or, optimistically, centuries. But, during this time, seeds eventually lose the ability to germinate. One explanation for this change is that the molecular machinery has gradually accumulated damage. When trying to assess the health of a seed lot, the standard method is to use a subset of seeds for a germination test, but these results are not straightforward to interpret. We hypothesized that accumulated damage at the molecular level could be quantified as an independent measure of seed lot health and chose to examine RNA. We showed that integrity of total RNA declines with storage time in seeds of many species. To show whether mRNA was similarly affected, we compared transcript integrity in 23-year-old and 2-year-old soybean seeds by sequencing full-length cDNA using MinION. In 23-year-old seeds, certain transcripts were only partially sequenced, and we confirmed that this was because of transcript fragmentation at random sites. We quantified transcript degradation for all transcripts and found that degradation increased with transcript length. This result supports the hypothesis that random damage accumulates at the molecular level. We now anticipate using the integrity of long transcripts to assess seed health over time. 

Transposable elements (TEs) are mobile DNA elements potentially able to move and multiply within genomes. TEs account for 40% of total size genome in the cultivated rice, Oryza sativa, and 10% in Arabidopsis thaliana and their insertion polymorphisms are responsible for most of the structural variations between varieties or ecotypes of these two species. Although large genomic datasets are available to study the diversity within these two species, most of these datasets consist in Illumina short read files and not in fully assembled genomes. To detect TE insertion polymorphisms (TIPs) in these large datasets, we developed a new software, TRACKPOSON and applied it to characterise TIPs for 31 families of retrotransposons in 3,000 cultivated rice varieties. We used DNA nanopore sequencing to unambiguously validate our in silico results. We further took advantage of cDNA nanopore sequencing to analyze structural variation within transcripts in rice and Arabidopsis and to improve transcriptome annotation with alternative splicing detection. Moreover, for the first time we could detect long reads corresponding to entire TE transcripts.

Complex genomes, including the human genome, contain ‘dark’ regions that standard short-read sequencing technologies do not adequately resolve, including protein-coding genes, leaving many variants that may be relevant to disease entirely overlooked. We systematically identified gene regions that are ‘dark by depth’ (few mappable reads), and others that are ‘camouflaged’ (ambiguous alignment). More than 100 protein-coding genes are 100% camouflaged using standard short-read sequencing. Many known disease-relevant genes are also camouflaged, including CR1, a top Alzheimer’s disease gene, and other disease-relevant genes include NEB, SMN1 and SMN2, and ARX. We further assessed how well long-read technologies resolve these regions, including 10x Genomics, PacBio’s Sequel, and Oxford Nanopore PromethION (Cliveome v. 3.0). We found that long-read technologies largely resolve the camouflaged gene regions, making it possible to identify mutations that may be important in human disease.

Various kinds of complex mutation (e.g. tandem-repeat expansion/contraction, homologous recombination, chromosome shattering, virus/transposon insertion) are known to cause diseases but have been neglected because they are hard to identify. I will describe our pipeline to find such mutations in patients. We perform whole-genome nanopore sequencing, find and group reads with structural differences from a reference genome, then de-prioritize differences shared by humans without the disease. We find most probable alignments between reads and reference, allowing for arbitrary rearrangements, based on probabilities of nucleotides, substitutions, insertions, and deletions. Our pipeline discovered the cause of neuronal intranuclear inclusion disease: expansion of a GGC tandem repeat in NOTCH2NLC. We can fully characterize complex congenital mutations caused by chromosome shattering. Some important properties of these rearrangements, such as sequence loss, are holistic: they are not present in any part of the rearrangement but are apparent only in the rearrangement as a whole.

The human transcriptome is highly diverse and complex, as evidenced by cell-type specific expression of unique transcript isoforms. In particular, transcripts derived from non-coding regions are the most qualitatively diverse class of genetic elements, encompassing over 70% of the genome. In contrast, about 80% of GWAS SNPs reside in non-coding regions, which suggests long non-coding RNAs may be the missing link, at least to some degree. I will present our recent work on high-resolution cDNA sequencing of non-coding regions associated with neuropsychiatric disorders using targeted sequencing on the PromethION. I will detail the discovery of new non-coding RNAs, mRNA isoforms, long-range exon dependencies, and how these relate to mental health and neurodegeneration. Given the apparent involvement of RNA modifications in neurological diseases, I will then segue into direct RNA sequencing and describe our in vitro strategy to train RNA base callers and detect modified RNA bases. This will include data from our recent preprint on detecting m6A in RNA with 90% accuracy. Finally, I will present how we maximise flow cell output by complementing an innovative RNA barcoding strategy and 'AI'. 

Oxford Nanopore sequencing technology has made it possible to sequence and de novo assemble plant genomes at relatively low cost with fast turn-around times. Continuous improvements in the technology as well as our optimization of DNA extraction, size selection and library preparation in combination with current long-read assemblers enable us to assemble larger plant genomes to a high-quality draft level. Using Hi-C we are further able to improve those high-quality contiguous genomes to chromosome-scale assemblies. This talk will highlight the progress we made in the past 3 years with this technology on the examples of the genomes of Solanum pennellii, Vinca minor, Physalis ixocarpa and Physalis alkekengi and also show how genes can be easily structurally annotated in de novo genome assemblies using the Oxford Nanopore RNA-seq technology. The low error-rate of these gene models derived from polished nanopore assemblies also allows for high-throughput functional annotation with the Mercator 4 pipeline.

Genome replication is a stochastic process whereby each cell exhibits different patterns of origin activation and replication fork movement. Despite this heterogeneity, replication is a remarkably stable process that works quickly and correctly over hundreds of thousands of iterations. Existing methods for measuring replication dynamics largely focus on how a population of cells behave on average, which precludes the detection of low probability errors that may have occurred in individual cells. These errors can have a severe impact on genome integrity, yet existing single-molecule methods, such as DNA combing, are too costly, low-throughput, and low-resolution to effectively detect them. We have created a method called D-NAscent that uses Oxford Nanopore sequencing to create high-throughput genome-wide maps of DNA replication dynamics in single molecules. I will discuss the informatics approach that our software uses, as well as questions pertaining to DNA replication and genome stability that our method is uniquely positioned to answer.

Single cell RNA-seq (scRNA-seq) is rapidly gaining favour for understanding biology. Long-read methods have great potential for scRNA-seq by facilitating the identification and quantification of gene isoforms, allowing cell specific variation in isoform expression and splicing to be characterised. Previous methods for transcriptome-wide long-read scRNA-seq have been limited by low numbers of cells and/or few reads per cell. We have developed an improved nanopore long-read scRNA-seq method that allows the profiling of hundreds of single-cells at high read-depth and demonstrate its use by profiling five human cancer cell lines. These results demonstrate the power of long-read sequencing to characterise gene and isoform expression in single cells. 

To catalogue and associate all forms of human genetic variation to health and disease, a new generation of genome sequencing and assembly technologies is required. However, current workflows for producing high-quality human genome assemblies have overall cost and production time bottlenecks that prohibit scaling to hundreds of individuals. We designed and evaluated an optimized PromethION-based workflow to produce near reference quality genome assemblies for the offsprings from ten parent-offspring trios. We demonstrate the production of long read, high-quality, and high-coverage genomes with a less than one-week total turnaround time from sample extraction to complete assembly, and a total projected cost of less than $10k per genome. To lower costs and improve quality we have developed three new tools: 1) Shasta - a nanopore de novo long read assembler that on a single compute node can produce complete human genomes in around 6 hours; 2) marginPolish - a new graphical model-based assembly polisher that improves on earlier methods in both cost and accuracy; and 3) HELEN - an RNN-based multi-task learning model that further refines the base and run-length prediction for each genomic position and produces state-of-the-art results. We evaluate the performance based on assembly accuracy, throughput/timing, and cost and demonstrate improvements relative to current best-of-breed in all areas. Recognizing that even 100kb reads are insufficient to scaffold through the most repetitive regions of the human genome, we augment this sequencing with a Hi-C long-range library to facilitate scaffolding and haplotype phasing.

Precise genome editing by the CRISPR/Cas9 system has proven to be ground-breaking in basic research.  Cas9 protein is increasingly being used for genome editing by direct transfection of an active guide RNA Cas9 ribonucleoprotein (RNP) complex into cells which introduce double-stranded breaks (DSBs) at targeted genomic loci. DSBs are repaired by endogenous cellular pathways such as non-homologous end joining (NHEJ) and homology-directed repair (HDR). Providing a ssDNA template during repair allows researchers to precisely introduce a desired mutation by utilizing the HDR pathway. However, rates of HDR are often low compared to NHEJ-mediated repair and analysis of large (>100 nt) insertions can be challenging.  Long read sequencing technology allows for a more comprehensive analysis of the outcome of large insertions or deletions created by CRISPR/Cas9 genome editing. Here, we use a target enrichment approach to selectively sequence a region of interest (ROI) around the CRISPR edited site to measure the rates of precise insertion by HDR.

The use of the portable MinION sequencer in plant pathology is rapidly increasing. Many studies have shown that the accuracy, portability and reduced time to result using the MinION are actively changing the way we do diagnostics and new diagnostic development for pests and diseases in agriculture. The first advantage of the MinION is clearly the ability to obtain rapid preliminary IDs of unknown pests and disease in the field. This was demonstrated recently by the Cassava Virus Action Project, taking just 4 hours to identify the virus present in symptomatic cassava plants in the field. The other advantage that can be overlooked is the opportunity to reduce the turn-around time to diagnosis for unknown pests and pathogens in a laboratory setting, as well as avoiding the need for expensive specialised equipment. While much has been made of the advances in in-field diagnostics, we wanted to answer the question of how does the data stack up in a laboratory setting when compared with other technologies? The small start-up costs and easy access to MinION sequencing, compared to other technologies makes it very attractive to plant virologists. Our team took a set of plant RNA samples from field pea with a known viral composition of a Potyvirus (Pea seed-borne mosaic virus - polyadenylated) and a Polerovirus (Turnip yellows virus – non polyadenylated), which we already had an Illumina data set for, and sequenced the samples using a cDNA and direct RNA kit on a MinION.  We compared downstream analyses performed with both the Illumina and MinION data. The results of our research suggest that not only is MinION suitable for rapid diagnostics in the laboratory and the field, but it is also useful in a wider research capacity.

Structural variants (SVs) are difficult to ascertain using short-read sequencing technology. As part of the Personalized OncoGenomics (POG) study, tumour and matched normal blood Illumina whole-genome sequencing was performed in patients with advanced cancers. We used Oxford Nanopore sequencing for validation and breakpoint resolution of four germline SVs in hereditary cancer genes: 1) ATM deletion, 2) NTHL1-TSC2-TRAF7 complex rearrangements, 3) IFT140-TSC2 inversion and 4) UIMC1-NSD1 complex rearrangements. The 12 breakpoints of these 4 SVs were seen in the nanopore data. Long-read sequencing was necessary for the resolution of SVs and corrected the initial interpretation in 3 out of 4 cases. Our results also showed the suspected IFT140-TSC2 large inversion to be a small intronic inverted duplication event that did not disrupt either gene. Our results suggest that short-read technology may not be sufficient for SVs assessment. Long-read sequencing technology may eventually be considered as an option for the detection and validation of clinically relevant germline SVs. 

Ultra-long DNA extraction and library prep protocols have generated sequencing reads of up to 2.3 Mb on MinION flow cells, with individual runs yielding multiple reads longer than 1 Mb and N50 values higher than 100 kb. Optimisation experiments are currently being carried out to increase flow cell yields and further improve read length statistics across all Oxford Nanopore platforms. Results shown will compare DNA extracted from a diverse range of organisms and sample types and include QC comparisons from different ultra-high molecular weight (ultra-HMW) extraction protocols including manual and automated approaches. We will also share progress updates comparing library preps from these samples run over Flongle, MinION and PromethION flow cells.

In light of widespread resurgence of the respiratory disease whooping cough, ongoing research aims to identify changes to the causative bacterium, Bordetella pertussis. B. pertussis is traditionally described as a highly clonal species at the single-base level, hence our research largely focusses on identifying differences between strains on a whole-genome scale. Long-read sequencing has enabled us to produce closed genome sequences for B. pertussis isolates on an unprecedented scale, allowing visualisation of extensive inter-strain genomic rearrangements. This work also led to the unexpected discovery of a second phenomenon: large duplications which are present in some recent isolates but not in the B. pertussis reference genome. Intriguingly, these duplications may be present in only a fraction of the cells of duplication-carrying strains. At London Calling 2019, I will discuss this developing story, including the essential role of long and ultra-long nanopore sequencing in proving the existence of the duplications and characterising variable populations, alongside continuing work to quantify the phenotypic effects of the duplications.

To ensure the safety of blood transfusions it is critical to match the blood type of both donor with the recipient. Current typing methods use monoclonal antibodies, however, reagents for rare blood groups are expensive, unavailable or unreliable. DNA-based identification of human blood groups has been used to overcome these limitations and its application has reduced rates of alloimmunisation in chronically transfused patients. While recent studies have shown the high degree of blood typing accuracy that can be achieved with modern high-throughput molecular techniques, structural variants and rare recombination events in the genome remain a source of error. Long range sequencing technologies can be leveraged to produce high quality haplotype reference sequences for the blood group encoding genes which can be used to improve current typing algorithms. 

The availability of substantially longer reads with the Oxford Nanopore approach opens new possibilities in many fields and explains the increasing use of the nanopore technology. To facilitate access and match storage as well as processing routines to the higher demand, we assembled Nanopype a modular, parallelized and easy-to-use pipeline to process the sequencing data from the raw signal output into standardized formats. Specifically, Nanopype facilitates the essential steps of base calling, quality control, and alignments, as well as various downstream applications by incorporating field-specific tools and complemented by custom utility scripts. To illustrate its application, we apply it to the assessment of short tandem repeats that have been implicated in neuropsychiatric disorders. Combined with a Cas12a-based enrichment strategy and the STRique package we show efficient targeting and quantification on raw signal level, as well as determination of the associated methylation status.

Complex environmental matrices, such as soil, sediment and excreta, are often synonymous with diverse microbial communities. Long read sequencing of DNA extracted from such communities can yield highly contiguous genomic data and provide information on both genetic composition and structure. However, DNA extracted from such matrices is often impure, fragmented and can potentially lack complete representation. Therefore, techniques such as isolation, enrichment and metagenomic assembly are used to answer questions on function and diversity. Here we present the sequencing and assembly of two environmental AMR harboring plasmids and one novel gc-rich genome isolated from the environment. Furthermore, we describe our exploration into the sequencing and analysis of long-range amplicon-based enrichment for AMR associated mobile genetic elements, and undertake metagenomic analysis of the community composition of two fractions of industrial anaerobic digesters. This has permitted us to investigate the evolution and selective drivers of AMR in the environment.

Infections caused by antimicrobial resistant bacterial pathogens are fast becoming an important global public health issue. Using next generation sequencing data of whole sediment and cultured fractions, our research group have identified wastewater treatment plants (WWTPs) as hotspots for the dissemination of antimicrobial resistance genes/bacteria (ARG/ARB) into the environment. Whilst WWTPs can remove up to 99.9% faecal coliforms, our results suggest that anaerobic digestors and the water treatment process positively select for ARG/ARB. The persistence of plasmid mediated ARGs outside of the host-associated system may play a compounding role in shaping the community-acquired resistome. The aim of our research is to understand the mechanisms of enzyme secretion in E. coli and determine why only ESBLs are in the exoproteome and no other beta-lactamases. Here we investigated the secretory mechanism of an ESBL-producing E. coli strain ST131 isolated from a UK water system. Strains of E. coli ST131 carrying multiple resistance genes, including blaCTX-M-15 (encoding extended spectrum beta-lactamase, ESBL), were isolated from the rivers downstream of WWTPs. We then quantified survival under prolonged anaerobic digestion in the presence and absence of selective antibiotics. We also confirmed if the gene was plasmid borne and studied the secretory mechanisms associated with all beta-lactamases in the genome. Here we present a method to rapidly sequence, assemble and undertake primary annotation of the bla genes carrying plasmid that are associated with our environmental E. coli ST131. The use of Oxford Nanopore long-read sequencing has permitted accurate de novo assembly and has helped further resolve the AMR genes location, composition, order, function and putative mechanism of transposition. Such assembly has been previously unachievable using our existing short-read sequence data set.

Setting up your first few nanopore sequencing runs are super exciting but far too often they end with disappointing yields for those of us who work with plants and other more challenging sample types. Frequently, the DNA extracted using standard kits and common solvent-based techniques are contaminated with substances that are incompatible with nanopore sequencing. I will share with you some methods I have found useful to prepare high quality DNA from plants, how to recognise what contaminants are present and some methods to get rid of them.  

There is an existing need for clinical tools that can be used to rapidly assess genomic variants and epigenetic changes at medically relevant genes. We have been using the CRISPR/Cas9 system for target-enrichment nanopore sequencing. We show the ability of this method to generate greater than 200X average coverage at 10 genomic loci (mean size 18kb) with a single MinION flow cell. We demonstrate that this high coverage data enables us to (1) profile DNA methylation patterns at cancer driver genes, (2) detect structural variations at known hot spots, and (3) survey for the presence of single nucleotide mutations. We demonstrate applications of this technique by examining the well-characterized GM12878 cell line as well as three breast cell lines (MCF-10A, MCF-7, MDA-MB-231) with varying tumorigenic potential as a model for cancer.

The accurate genotyping of CYP2D6 is hindered by the very polymorphic nature of the gene, high homology with its pseudogene CYP2D7, and the occurrence of structural variations. Using the GridION nanopore sequencer, we sequenced 32 samples covering various haplotypes of CYP2D6, including four samples with gene duplication, over two sequencing runs. The haplotypes of 26 samples could be matched accurately to known alleles or subvariants, while the remaining 6 samples had either novel variants or variant patterns not matched to the current PharmVar CYP2D6 haplotype database. Small insertions/deletions associated with several key haplotypes were detected accurately, and five novel variants not yet catalogues in PharmVar were reported. Allele duplication could be determined by analyzing the allelic balance between the sample haplotypes. Nanopore sequencing of CYP2D6 offers a high throughput method for genotyping, accurate haplotyping, and detection of new variants and duplicated alleles.