Nanopore-based Sequencing Research Articles

Detection of Nuclear RNA Decay Intermediates Using a Modified Oxford Nanopore RNA Sequencing Strategy.

The nuclear RNA exosome complex is crucial for noncoding RNA processing and RNA quality control in the nucleus. Identifying substrates and intermediates of RNA decay pathways, such as those mediated by the exosome complex using Oxford Nanopore sequencing can be difficult in part because a simple method to detect them has been lacking and also because some of these RNAs lack abundant poly(A) tails which are required for Oxford Nanopore-based sequencing. Here we describe an Oxford nanopore-based approach which can be used to identify long reads corresponding to precursors and products of nuclear exosome processing. We are able to observe accumulation of unprocessed snoRNAs, cleavage products of the yeast nuclear RNase III endonuclease Rnt1p when the nuclear exosome component Rrp6p is inactivated.

Complete genome characterization by nanopore sequencing of rotaviruses A, B, and C circulating on large-scale pig farms in Russia.

Rotaviruses are the major etiological agents of gastroenteritis and diarrheal outbreaks in plenty of mammalian species. The genus Rotavirus is highly diverse and currently comprises nine genetically distinct species, and four of them (A, B, C, and H) are common for humans and pigs. There is a strong necessity to comprehend phylogenetic relationships among rotaviruses from different host species to assess interspecies transmission, specifically between humans and livestock. To reveal the genetic origin of rotaviruses from Russian pig farms, nanopore-based metagenomic sequencing was performed on the PCR-positive specimens. Samples were selected among the cases submitted to routine diagnostic or monitoring studies to the Laboratory of Biochemistry and Molecular Biology of "Federal Scientific Center VIEV" (Moscow, Russia). The selected positive samples were genotyped using nanopore sequencing method. Five porcine RVA isolates were completely sequenced, and genotype analysis revealed various porcine G/P genogroups: G2, G3, G4, G5, G11 and P[6], P[7], P[13], P[23], P[27] with a typical backbone constellation I5-R1-C1-M1-A8-N1-T1/7-E1-H1. The RVB isolate was detected in combination with RVA in a rectal swab from a diseased pig in Krasnoyarsk Krai. It was characterized by the following genogroups: G15-P[X]-I11-R4-C4-M4-A8-N10-T4-E4-H7. The first complete porcine RVC genome from Russia was obtained with genomic constellation G6-P[5]-I14-R1-C1-M1-A7-N9-T6-E1-H1, and the phylogenetic analysis revealed putative novel genotype group for the VP6 gene-I14. Additionally, the first porcine kobuvirus isolate from Russia was phylogenetically characterized. The applied nanopore sequencing method successfully genotyped the RV isolates and additionally revealed co-circulated species. The study demonstrates high genetic variability of Russian RVA isolates in VP4/VP7 genes and phylogenetically describes local RVB and RVC. Complete characterization of genomic segments is a crucial methodology in tracing the rotavirus's evolution and evaluating interspecies transmissions.

Nanopore-Based Sequencing of the Full-Length Transcriptome of Male and Female Cleavage-Stage Embryos of the Chinese Mitten Crab (Eriocheir sinensis)

The cleavage stage plays a crucial role in embryo development, characterized by a swift surge in cell proliferation alongside the accurate genetic material transmission to offspring. To delve into the characteristics of sex development during the cleavage stage of embryos, we generated the full-length transcriptome of Eriocheir sinensis male and female cleavage-stage embryos using Oxford Nanopore Technologies (ONT). Notably, this investigation represents the first sequencing effort distinguishing between genders in E. sinensis embryos. In the transcriptome structure analysis, male and female cleavage-stage embryos, while not clustered, exhibited a comparable frequency of alternative splicing (AS) occurrences. We also successfully identified 2875 transcription factors (TFs). The quantitative analysis showed the top 150 genes, in which the highly expressed genes in male embryos predominantly related to protein synthesis and metabolism. Further investigation unveiled 500 differentially expressed genes (DEGs), of which 7 male-biased ribosomal protein genes (RPGs) were particularly noteworthy and further confirmed. These analyses suggest that there may be a more active protein synthesis process in male E. sinensis cleavage-stage embryos. Furthermore, among the 2875 identified TFs, we predicted that 18 TFs could regulate the differentially expressed RPGs, with most TFs belonging to the zf-C2H2 and Homeobox families, which are crucial for embryonic development. During the cleavage stage of E. sinensis, the differential RPGs between genders were intricately linked to energy metabolism. We proposed that these RPGs exert regulatory effects on gene expression in E. sinensis, thereby regulating the difference of development between male and females. Our research sheds light on the developmental mechanisms of E. sinensis during the embryo stage and establishes a groundwork for a deeper understanding of sex development in E. sinensis. The results also provide comprehensive full-length transcriptome data for future gene expression and genetic studies in E. sinensis.

Generation and analysis of a mouse multitissue genome annotation atlas.

Generating an accurate and complete genome annotation for an organism is complex because the cells within each tissue can express a unique set of transcript isoforms from a unique set of genes. A comprehensive genome annotation should contain information on what tissues express what transcript isoforms at what level. This tissue-level isoform information can then inform a wide range of research questions as well as experiment designs. Long-read sequencing technology combined with advanced full-length cDNA library preparation methods has now achieved throughput and accuracy where generating these types of annotations is achievable. Here, we show this by generating a genome annotation of the mouse (Mus musculus). We used the nanopore-based R2C2 long-read sequencing method to generate 64 million highly accurate full-length cDNA consensus reads-averaging 5.4 million reads per tissue for a dozen tissues. Using the Mandalorion tool, we processed these reads to generate the Tissue-level Atlas of Mouse Isoforms which is available as a trackhub for the UCSC Genome Browser and contains at least one full-length isoform for the vast majority of expressed genes in each tissue.

Investigating the Quantification Capabilities of a Nanopore-Based Sequencing Platform for Food Safety Application via External Standards of Lambda DNA and Lambda Spiked Beef.

Six hundred million cases of disease and roughly 420,000 deaths occur globally each year due to foodborne pathogens. Current methods to screen and identify pathogens in swine, poultry, and cattle products include immuno-based techniques (e.g., immunoassay integrated biosensors), molecular methods (e.g., DNA hybridization and PCR assays), and traditional culturing. These methods are often used in tandem to screen, quantify, and characterize samples, prolonging real-time comprehensive analysis. Next-generation sequencing (NGS) is a relatively new technology that combines DNA-sequencing chemistry and bioinformatics to generate and analyze large amounts of short- or long-read DNA sequences and whole genomes. The goal of this project was to evaluate the quantitative capabilities of the real-time NGS Oxford Nanopore Technologies' MinION sequencer through a shotgun-based sequencing approach. This investigation explored the correlation between known amounts of the analyte (lambda DNA as a pathogenic bacterial surrogate) with data output, in both the presence and absence of a background matrix (Bos taurus DNA). A positive linear correlation was observed between the concentration of analyte and the amount of data produced, number of bases sequenced, and number of reads generated in both the presence and absence of a background matrix. In the presence of bovine DNA, the sequenced data were successfully mapped to the NCBI lambda reference genome. Furthermore, the workflow from pre-extracted DNA to target identification took less than 3 h, demonstrating the potential of long-read sequencing in food safety as a rapid method for screening, identification, and quantification.

Detecting m6A RNA modification from nanopore sequencing using a semisupervised learning framework.

Direct nanopore-based RNA sequencing can be used to detect posttranscriptional base modifications, such as N6-methyladenosine (m6A) methylation, based on the electric current signals produced by the distinct chemical structures of modified bases. A key challenge is the scarcity of adequate training data with known methylation modifications. We present Xron, a hybrid encoder-decoder framework that delivers a direct methylation-distinguishing basecaller by training on synthetic RNA data and immunoprecipitation (IP)-based experimental data in two steps. First, we generate data with more diverse modification combinations through in silico cross-linking. Second, we use this data set to train an end-to-end neural network basecaller followed by fine-tuning on IP-based experimental data with label smoothing. The trained neural network basecaller outperforms existing methylation detection methods on both read-level and site-level prediction scores. Xron is a standalone, end-to-end m6A-distinguishing basecaller capable of detecting methylated bases directly from raw sequencing signals, enabling de novo methylome assembly.

Nanopore-based full-length transcriptome sequencing of the skin in Pseudopleuronectes yokohamae identifies novel antimicrobial peptide genes

The marbled flounder (Pseudopleuronectes yokohamae) is highly esteemed for its exceptional nutritional value and delicious taste. However, this species has extremely limited transcriptome data, which can offer priceless information for disease protection. In the study, the skin transcriptomic sequencing of P. yokohamae revealed 7.72 GB of clean data using the Nanopore sequencing platform. The results revealed 30,498 transcripts of functional annotations in the P. yokohamae transcriptome. All transcripts were searched in eight functional databases. A total of 10,337 ORFs were obtained, of which 6081 complete ORFs accounted for 58.83% of all predicted CDS. Moreover, 10,195 SSRs were detected. Meanwhile, the non-pecific immunity pathways were investigated for better understanding of the immunological reaction in P. yokohamae, and seven innate immune pathways were identified. The innate-immune related genes were highly expressed in the NOD-like receptor signaling pathway, followed by the C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and Cytosolic DNA-sensing pathway. In this study, four families of antimicrobial peptides (AMPs) in P. yokohamae were analysed for the first time, including piscidins, hepcidins, liver-expressed antimicrobial peptide and defensins. Seven AMPs, including Pypleurocidin-like WF3, Pypleurocidin-like WFX, Pyhepcidin 1, Pyhepcidin-like 1, PyLEAP-2, Pybeta-defensin and Pybeta-defensin-like 1, were further identified. The seven AMPs showed a highly identity in their cDNA and genomic structures and an inducible expression pattern preferable to skin in response to pathogens. The transcriptomic data and investigation of AMPs from P. yokohamae promote a deeper awareness of fish mucosal immunity and provide information in the prevention of fish diseases.

Nanopore-based full-length transcriptome sequencing for understanding the underlying molecular mechanisms of rapid and slow progression of diabetes nephropathy.

Diabetic nephropathy (DN) has been a major factor in the outbreak of end-stage renal disease for decades. As the underlying mechanisms of DN development remains unclear, there is no ideal methods for the diagnosis and therapy. We aimed to explore the key genes and pathways that affect the rate progression of DN. Nanopore-based full-length transcriptome sequencing was performed with serum samples from DN patients with slow progression (DNSP, n = 5) and rapid progression (DNRP, n = 6). Here, transcriptome proclaimed 22,682 novel transcripts and obtained 45,808 simple sequence repeats, 1,815 transcription factors, 5,993 complete open reading frames, and 1,050 novel lncRNA from the novel transcripts. Moreover, a total of 341 differentially expressed transcripts (DETs) and 456 differentially expressed genes (DEGs) between the DNSP and DNRP groups were identified. Functional analyses showed that DETs mainly involved in ferroptosis-related pathways such as oxidative phosphorylation, iron ion binding, and mitophagy. Moreover, Functional analyses revealed that DEGs mainly involved in oxidative phosphorylation, lipid metabolism, ferroptosis, autophagy/mitophagy, apoptosis/necroptosis pathway. Collectively, our study provided a full-length transcriptome data source for the future DN research, and facilitate a deeper understanding of the molecular mechanisms underlying the differences in fast and slow progression of DN.

Units containing telomeric repeats are prevalent in subtelomeric regions of a Mesorhabditis isolate collected from the Republic of Korea.

Mesorhabditis is known for its somatic genome being only a small portion of the germline genome due to programmed DNA elimination. This phenotype may be associated with the maintenance of telomeres at the ends of fragmented somatic chromosomes. To comprehensively investigate the telomeric regions of Mesorhabditis nematodes at the sequence level, we endeavored to collect a Mesorhabditis nematode in the Republic of Korea and acquire its highly contiguous genome sequences. We isolated a Mesorhabditis nematode and assembled its 108-Mb draft genome using both 6.3Gb (53 ×) of short-read and 3.0Gb (25 × , N50 = 5.7kb) of nanopore-based long-read sequencing data. Our genome assembly exhibits comparable quality to the public genome of Mesorhabditis belari in terms of contiguity and evolutionary conserved genes. Unexpectedly, our Mesorhabditis genome has many more interstitial telomeric sequences (ITSs), specifically subtelomeric ones, compared to the genomes of Caenorhabditis elegans and M. belari. Moreover, several subtelomeric sequences containing ITSs had 4-26 homologous sequences, implying they are highly repetitive. Based on this highly repetitive nature, we hypothesize that subtelomeric ITSs might have accumulated through the action of transposable elements containing ITSs. It still remains elusive whether these ITS-containing units are associated with programmed DNA elimination, but they may facilitate new telomere formation after DNA elimination. Our genomic resources for Mesorhabditis can aid in understanding how its distinct phenotypes have evolved.

Average Nucleotide Identity and Digital DNA-DNA Hybridization Analysis Following PromethION Nanopore-Based Whole Genome Sequencing Allows for Accurate Prokaryotic Typing.

Whole-genome sequencing (WGS) is revolutionizing clinical bacteriology. However, bacterial typing remains investigated by reference techniques with inherent limitations. This stresses the need for alternative methods providing robust and accurate sequence type (ST) classification. This study optimized and evaluated a GridION nanopore sequencing protocol, adapted for the PromethION platform. Forty-eight Escherichia coli clinical isolates with diverse STs were sequenced to assess two alternative typing methods and resistance profiling applications. Multi-locus sequence typing (MLST) was used as the reference typing method. Genomic relatedness was assessed using Average Nucleotide Identity (ANI) and digital DNA-DNA Hybridization (DDH), and cut-offs for discriminative strain resolution were evaluated. WGS-based antibiotic resistance prediction was compared to reference Minimum Inhibitory Concentration (MIC) assays. We found ANI and DDH cut-offs of 99.3% and 94.1%, respectively, which correlated well with MLST classifications and demonstrated potentially higher discriminative resolution than MLST. WGS-based antibiotic resistance prediction showed categorical agreements of ≥ 93% with MIC assays for amoxicillin, ceftazidime, amikacin, tobramycin, and trimethoprim-sulfamethoxazole. Performance was suboptimal (68.8-81.3%) for amoxicillin-clavulanic acid, cefepime, aztreonam, and ciprofloxacin. A minimal sequencing coverage of 12× was required to maintain essential genomic features and typing accuracy. Our protocol allows the integration of PromethION technology in clinical laboratories, with ANI and DDH proving to be accurate and robust alternative typing methods, potentially offering superior resolution. WGS-based antibiotic resistance prediction holds promise for specific antibiotic classes.

Escherichia coli novel sequence type 11873 harbours a new CTX-M-15–carrying multidrug resistance type 1/2 hybrid IncC plasmid

ObjectiveThe aim of the current study was to determine the genomic map of the resistance genes of two CTX-M-15–carrying Escherichia coli strains belonging to novel sequence type (ST) 11873. Complete, closed genome sequences of the E. coli strains were obtained by applying a combination of short-read Illumina and long-read Oxford Nanopore-based sequencing. MethodsIsolation of E. coli was performed using ECC CHROMagar and antibiotic sensitivity patterns were determined using Sensititre EUVSEC plates. Whole-genome sequencing was performed for two E. coli strains (3–338 and 5–325) using Illumina MiSeq- and Oxford Nanopore MinION-based sequencing. ResultsThe complete genome of strain 3–338 (GenBank accession no. CP130007–17) was assembled into a circular chromosome of 4.65 Mb and 10 plasmids (between 2 and 148 kb). Strain 5–325 (CP130018–27) exhibited a circular chromosome of 4.7 Mb and 9 plasmids (between 2 and 148 kb). Both strains carried an identical type 1/2 hybrid IncC plasmid (∼148 kb) harbouring multiple antibiotic resistance genes (ARGs), including blaCTX−M-15, blaOXA-1, blaTEM-1, qnrS1, sul2, aphA1, aacC2, mph(A) and floR. This plasmid also carried heavy metal resistance genes, such as chrA and arsR. Strain 5–325 carried an additional IncFIB plasmid (∼78 kb) harbouring additional ARGs, including blaTEM-1, qnrS1, tet(A), dfrA14, sul2, strA and strB. ConclusionsOur study shows the emergence of a CTX-M-15–carrying type 1/2 hybrid IncC plasmid in novel E. coli ST11873. These findings emphasise the need for population-based sewage surveillance for understanding the prevalence of antibiotic resistance in pathogens in order to mitigate the further spread of such resistance factors.

A Comparison of Structural Variant Calling from Short-Read and Nanopore-Based Whole-Genome Sequencing Using Optical Genome Mapping as a Benchmark.

The identification of structural variants (SVs) in genomic data represents an ongoing challenge because of difficulties in reliable SV calling leading to reduced sensitivity and specificity. We prepared high-quality DNA from 9 parent-child trios, who had previously undergone short-read whole-genome sequencing (Illumina platform) as part of the Genomics England 100,000 Genomes Project. We reanalysed the genomes using both Bionano optical genome mapping (OGM; 8 probands and one trio) and Nanopore long-read sequencing (Oxford Nanopore Technologies [ONT] platform; all samples). To establish a "truth" dataset, we asked whether rare proband SV calls (n = 234) made by the Bionano Access (version 1.6.1)/Solve software (version 3.6.1_11162020) could be verified by individual visualisation using the Integrative Genomics Viewer with either or both of the Illumina and ONT raw sequence. Of these, 222 calls were verified, indicating that Bionano OGM calls have high precision (positive predictive value 95%). We then asked what proportion of the 222 true Bionano SVs had been identified by SV callers in the other two datasets. In the Illumina dataset, sensitivity varied according to variant type, being high for deletions (115/134; 86%) but poor for insertions (13/58; 22%). In the ONT dataset, sensitivity was generally poor using the original Sniffles variant caller (48% overall) but improved substantially with use of Sniffles2 (36/40; 90% and 17/23; 74% for deletions and insertions, respectively). In summary, we show that the precision of OGM is very high. In addition, when applying the Sniffles2 caller, the sensitivity of SV calling using ONT long-read sequence data outperforms Illumina sequencing for most SV types.

Direct RNA sequencing of astronaut blood reveals spaceflight-associated m6A increases and hematopoietic transcriptional responses

The advent of civilian spaceflight challenges scientists to precisely describe the effects of spaceflight on human physiology, particularly at the molecular and cellular level. Newer, nanopore-based sequencing technologies can quantitatively map changes in chemical structure and expression at single molecule resolution across entire isoforms. We perform long-read, direct RNA nanopore sequencing, as well as Ultima high-coverage RNA-sequencing, of whole blood sampled longitudinally from four SpaceX Inspiration4 astronauts at seven timepoints, spanning pre-flight, day of return, and post-flight recovery. We report key genetic pathways, including changes in erythrocyte regulation, stress induction, and immune changes affected by spaceflight. We also present the first m6A methylation profiles for a human space mission, suggesting a significant spike in m6A levels immediately post-flight. These data and results represent the first longitudinal long-read RNA profiles and RNA modification maps for each gene for astronauts, improving our understanding of the human transcriptome’s dynamic response to spaceflight.

Rapid genotyping of Toxoplasma gondii isolates via Nanopore-based multi-locus sequencing

Toxoplasma gondii is an obligate intracellular parasite associated with severe disease, especially in the immunosuppressed. It is also a cause of congenital malformation and abortion in both animals and humans and is considered one of the most important foodborne pathogens worldwide with different strains showing variable distribution and differing pathogenicity. Thus, strain-level differentiation of T. gondii isolates is an essential asset in the understanding of parasite’s diversity, geographical distribution, epidemiology and health risk. Here, we designed and implemented an Oxford Nanopore MinION protocol to analyse genomic sequence variation including single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (InDel’s) of four different genomic loci, part of protein coding genes SAG2, SAG3, ROP17 and ROP21. This method provided results with the sequencing depth necessary for accurate differentiation of T. gondii strains and represents a rapid approach compared to conventional techniques which we further validated against environmental samples isolated from wild wood mice. In summary, multi-locus sequence typing (MLST) of both highly conserved and more polymorphic areas of the genome, provided robust data for strain classification in a platform ready for further adaption for other strains and pathogens.

Comparison of CcrM-dependent methylation in Caulobacter crescentus and Brucella abortus by nanopore sequencing.

Bacteria rely on DNA methylation for restriction-modification systems and epigenetic control of gene expression. Here, we use direct detection of methylated bases by nanopore sequencing to monitor global DNA methylation in Alphaproteobacteria, where use of this technique has not yet been reported. One representative of this order, Caulobacter crescentus, relies on DNA methylation to control cell cycle progression, but it is unclear whether other members of this order, such as Brucella abortus, depend on the same systems. We addressed these questions by first measuring CcrM-dependent DNA methylation in Caulobacter and showing excellent correlation between nanopore-based detection and previously published results. We then directly measure the impact of Lon-mediated CcrM degradation on the epigenome, verifying that loss of Lon results in pervasive methylation. We also show that the AlkB demethylase has no global impact on DNA methylation during normal growth. Next, we report on the global DNA methylation in B. abortus for the first time and find that CcrM-dependent methylation is reliant on Lon but impacts the two chromosomes differently. Finally, we explore the impact of the MucR transcription factor, known to compete with CcrM methylation, on the Brucella methylome and share the results with a publicly available visualization package. Our work demonstrates the utility of nanopore-based sequencing for epigenome measurements in Alphaproteobacteria and reveals new features of CcrM-dependent methylation in a zoonotic pathogen.IMPORTANCEDNA methylation plays an important role in bacteria, maintaining genome integrity and regulating gene expression. We used nanopore sequencing to directly measure methylated bases in Caulobacter crescentus and Brucella abortus. In Caulobacter, we showed that stabilization of the CcrM methyltransferase upon loss of the Lon protease results in prolific methylation and discovered that the putative methylase AlkB is unlikely to have a global physiological effect. We measured genome-wide methylation in Brucella for the first time, revealing a similar role for CcrM in cell-cycle methylation but a more complex regulation by the Lon protease than in Caulobacter. Finally, we show how the virulence factor MucR impacts DNA methylation patterns in Brucella.

Precise Structural Analysis of Neutral Glycans Using Aerolysin Mutant T240R Nanopore.

Glycans play vital roles in nearly all life processes of multicellular organisms, and understanding these activities is inseparable from elucidating the biological significance of glycans. However, glycan research has lagged behind that of DNA and protein due to the challenges posed by structural heterogeneity and isomerism (i.e., structures with equal molecular weights) the lack of high-efficiency structural analysis techniques. Nanopore technology has emerged as a sensitive single-molecule biosensor, shining a light on glycan analysis. However, a significant number of glycans are small and uncharged, making it challenging to elicit identifiable nanopore signals. Here we introduce a R-binaphthyl tag into glycans, which enhances the cation-π interaction between the derivatized glycan molecules and the nanopore interface, enabling the detection of neutral glycans with an aerolysin nanopore. This approach allows for the distinction of di-, tri-, and tetrasaccharides with monosaccharide resolution and has the potential for group discrimination, the monitoring of enzymatic transglycosylation reactions. Notably, the aerolysin mutant T240R achieves unambiguous identification of six disaccharide isomers, trisaccharide and tetrasaccharide linkage isomers. Molecular docking simulations reveal that multiple noncovalent interactions occur between residues R282, K238, and R240 and the glycans and R-binaphthyl tag, significantly slowing down their translocation across the nanopore. Importantly, we provide a demonstration of the kinetic translocation process of neutral glycan isomers, establishing a solid theoretical foundation for glycan nanopore analysis. The development of our technology could promote the analysis of glycan structural isomers and has the potential for nanopore-based glycan structural determination and sequencing.

Long-Read Nanopore-Based Sequencing of Anelloviruses.

Routinely used metagenomic next-generation sequencing (mNGS) techniques often fail to detect low-level viremia (<104 copies/mL) and appear biased towards viruses with linear genomes. These limitations hinder the capacity to comprehensively characterize viral infections, such as those attributed to the Anelloviridae family. These near ubiquitous non-pathogenic components of the human virome have circular single-stranded DNA genomes that vary in size from 2.0 to 3.9 kb and exhibit high genetic diversity. Hence, species identification using short reads can be challenging. Here, we introduce a rolling circle amplification (RCA)-based metagenomic sequencing protocol tailored for circular single-stranded DNA genomes, utilizing the long-read Oxford Nanopore platform. The approach was assessed by sequencing anelloviruses in plasma drawn from people who inject drugs (PWID) in two geographically distinct cohorts. We detail the methodological adjustments implemented to overcome difficulties inherent in sequencing circular genomes and describe a computational pipeline focused on anellovirus detection. We assessed our protocol across various sample dilutions and successfully differentiated anellovirus sequences in conditions simulating mixed infections. This method provides a robust framework for the comprehensive characterization of circular viruses within the human virome using the Oxford Nanopore.

Advancing pathogen surveillance by nanopore sequencing and genotype characterization of Acheta domesticus densovirus in mass-reared house crickets

Rapid and reliable detection of pathogens is crucial to complement the growing industry of mass-reared insects, in order to safeguard the insect colonies from outbreak of diseases, which may cause significant economic loss. Current diagnostic methods are mainly based on conventional PCR and microscopic examination, requiring prior knowledge of disease symptoms and are limited to identifying known pathogens. Here, we present a rapid nanopore-based metagenomics approach for detecting entomopathogens from the European house cricket (Acheta domesticus). In this study, the Acheta domesticus densovirus (AdDV) was detected from diseased individuals using solely Nanopore sequencing. Virus reads and genome assemblies were obtained within twenty-four hours after sequencing. Subsequently, due to the length of the Nanopore reads, it was possible to reconstruct significantly large parts or even the entire AdDV genome to conduct studies for genotype identification. Variant analysis indicated the presence of three AdDV genotypes within the same house cricket population, with association to the vital status of the diseased crickets. This contrast provided compelling evidence for the existence of non-lethal AdDV genotypes. These findings demonstrated nanopore-based metagenomics sequencing as a powerful addition to the diagnostic tool kit for routine pathogen surveillance and diagnosis in the insect rearing industry.

Abstract 2928: Solving the cancer mutation conundrum: A single cell, massively parallel approach for cancer mutation discovery, genome modelling and functional characterization

Abstract Large scale genomic studies are cataloguing tens of thousands of cancer mutations across a wide spectrum of tumor types. Conventional DNA sequencing methods have limitations in terms of identify mutations at high resolution among single cells. Moreover, determining cancer mutations’ phenotype and function remains an enormous challenge - most mutations are assigned to the category of variants of unknown significance. In-silico functional predictions of cancer mutations are frequently used as a stopgap solution. However, these computational methods lack the ability to make refined biological and functional predictions. We demonstrated a new single cell experimental strategy that accelerates the discovery of cancer mutations and their characterization. This genomic technology framework does the following: (1) identify disease-related mutations at the resolution of individual cells from tumors or cell lines; (2) genome engineer these candidate mutations into cellular experimental systems for further biological characterization. First, we developed a single cell approach to identify cancer somatic mutations and even gene fusion rearrangements found in coding regions of mRNAs. Primary tumors are processed into single cell RNA-seq libraries and analyzed with nanopore long read sequencing. Across thousands of cells from primary tumors, we identified single cell mutations from the nanopore long reads that covered cancer gene transcripts. This approach allows us to even identify cancer rearrangements at single cell resolution. Next, we used short-read single cell transcriptomes to characterize the cell type and determine the gene expression characteristics of those cells with mutations. For single cell phenotypic studies, we developed a highly multiplexed technology using CRISPR base editors to directly engineer hundreds of mutations into their original genes among single cells and determine their transcriptional phenotype. Among specific individual cells, nanopore-based long-read sequencing identifies these modelled mutations directly from a target’s transcript sequence. Then, we integrate this single cell mutation data with the short-read transcriptome profile from the same individual cell. Therefore, we determine both the genotype and phenotype of a given engineered mutation from an individual cell. In summary, our work demonstrates a new, direct and highly scalable method for identifying, modelling and functionally assessing cancer mutation phenotype at single cell resolution. This approach enables the broader assessment of cancer mutations impact on tumor biology and clinical ramifications. Citation Format: Susan M. Grimes, Heonseok Kim, Sharmili Roy, Anuja Sathe, Billy T. Lau, Hanlee P. Ji. Solving the cancer mutation conundrum: A single cell, massively parallel approach for cancer mutation discovery, genome modelling and functional characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2928.

Abstract 5049: Detecting chromosomal copy number variations and point mutations in Glioma using a single assay; sparing tissue while significantly reducing testing time and cost

Abstract We aimed to develop a single streamlined assay to detect all Glioma biomarkers. The current diagnosis of Gliomas incorporates molecular biomarkers that include whole chromosomal copy number variations (CNVs) and single nucleotide variations (SNVs). Testing CNVs and SNVs are traditionally done using different large platforms and are performed in large genomic facilities that have the suitable infrastructure. This creates delays in testing, where samples undergo lengthy referrals and the tissue is often split between institutions, which also adds substantially to the costs. We have previously used Nanopore sequencing to develop an assay to detect IDH SNVs from formalin-fixed paraffin-embedded (FFPE) tissue. Nanopore sequencing platform uses small inexpensive tools and can test for both CNVs and SNVs. However, combining both targets efficiently in FFPE tissue remains a challenge. While nanopore whole genome sequencing can detect CNVs, it does not allow for enough coverage to accurately detect small SNVs. Also, raw FFPE DNA is not compatible with the nanopore technology. To fix both challenges we used a PCR based approach that allows enrichment of the SNV targets, as well as creates new nanopore compatible DNA copies. To counteract the bias that PCR creates with uneven amplification, we used a SNP based method to call chromosomal gains and losses. Statistical analysis of probability showed that targeting 15 heterozygous SNPs per chr arm is sufficient for accurate CNV detection (error rate &amp;lt;0.1%). A total of 57 amplicons targeting CNVs on Chr 1,7, 10, 19, and EGFR gene; and IDH1 and IDH2 SNVs were pooled together in one assay and tested on 4 Glioma samples. Variant calling was performed with a custom Python script. All CNV targets were accurately detected on all samples. Losses in chr 1p, 19q and chr 10 were evident with a loss of heterozygosity (LOH) pattern on the SNP analysis. The gain in chromosome 7 was also observed as an allele gain 2:1 SNP pattern on the corresponding samples. The SNV statuses of these samples were also accurately detected when compared to reference results. Finally, the SNV and CNV calling analysis were streamlined using custom shell script, further reducing total turnaround time (including sequencing and data analysis) to less than a day for the entire batch of samples. Initial cost analysis shows &amp;lt;50% of the traditional testing costs for the assay. This work is the first to develop a streamlined single test to detect CNVs and SNVs in Gliomas using FFPE DNA on the nanopore sequencing platform. The shorter testing time, streamlined workflow, and low assay/capital cost could positively influence the care of brain cancer patients. Notably, nanopore-based sequencing tools can also be implemented in other types of tumor diagnosis, especially by smaller labs, helping to overcome many of the existing diagnostic challenges. Citation Format: Mashiat L. Mimosa, Jared T. Simpson, Karel Boissinot, Mora Tiab, Ramzi Fattouh, Rola M. Saleeb. Detecting chromosomal copy number variations and point mutations in Glioma using a single assay; sparing tissue while significantly reducing testing time and cost [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5049.