Metastasizing aneurysmal dermatofibroma initially diagnosed as angiosarcoma confirmed by CD63::PRKCD fusion gene detection with nanopore sequencing.

Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.

Haplotype determination of the Bombyx mori nucleopolyhedrovirus by Nanopore sequencing and linkage of single nucleotide variants.

Naturally occurring isolates of baculoviruses, such as the Bombyx mori nucleopolyhedrovirus (BmNPV), usually consist of numerous genetically different haplotypes. Deciphering the different haplotypes of such isolates is hampered by the large size of the dsDNA genome, as well as the short read length of next generation sequencing (NGS) techniques that are widely applied for baculovirus isolate characterization. In this study, we addressed this challenge by combining the accuracy of NGS to determine single nucleotide variants (SNVs) as genetic markers with the long read length of Nanopore sequencing technique. This hybrid approach allowed the comprehensive analysis of genetically homogeneous and heterogeneous isolates of BmNPV. Specifically, this allowed the identification of two putative major haplotypes in the heterogeneous isolate BmNPV-Ja by SNV position linkage. SNV positions, which were determined based on NGS data, were linked by the long Nanopore reads in a Position Weight Matrix. Using a modified Expectation-Maximization algorithm, the Nanopore reads were assigned according to the occurrence of variable SNV positions by machine learning. The cohorts of reads were de novo assembled, which led to the identification of BmNPV haplotypes. The method demonstrated the strength of the combined approach of short- and long-read sequencing techniques to decipher the genetic diversity of baculovirus isolates.

A comparison between full-length 16S rRNA Oxford nanopore sequencing and Illumina V3-V4 16S rRNA sequencing in head and neck cancer tissues.

Describing the microbial community within the tumour has been a key aspect in understanding the pathophysiology of the tumour microenvironment. In head and neck cancer (HNC), most studies on tissue samples have only performed 16S rRNA short-read sequencing (SRS) on V3-V5 region. SRS is mostly limited to genus level identification. In this study, we compared full-length 16S rRNA long-read sequencing (FL-ONT) from Oxford Nanopore Technology (ONT) to V3-V4 Illumina SRS (V3V4-Illumina) in 26 HNC tumour tissues. Further validation was also performed using culture-based methods in 16 bacterial isolates obtained from 4 patients using MALDI-TOF MS. We observed similar alpha diversity indexes between FL-ONT and V3V4-Illumina. However, beta-diversity was significantly different between techniques (PERMANOVA - R2 = 0.131, p < 0.0001). At higher taxonomic levels (Phylum to Family), all metrics were more similar among sequencing techniques, while lower taxonomy displayed more discrepancies. At higher taxonomic levels, correlation in relative abundance from FL-ONT and V3V4-Illumina were higher, while this correlation decreased at lower levels. Finally, FL-ONT was able to identify more isolates at the species level that were identified using MALDI-TOF MS (75% vs. 18.8%). FL-ONT was able to identify lower taxonomic levels at a better resolution as compared to V3V4-Illumina 16S rRNA sequencing.

Nm-Nano: a machine learning framework for transcriptome-wide single-molecule mapping of 2´-O-methylation (Nm) sites in nanopore direct RNA sequencing datasets.

2´-O-methylation (Nm) is one of the most abundant modifications found in both mRNAs and noncoding RNAs. It contributes to many biological processes, such as the normal functioning of tRNA, the protection of mRNA against degradation by the decapping and exoribonuclease (DXO) protein, and the biogenesis and specificity of rRNA. Recent advancements in single-molecule sequencing techniques for long read RNA sequencing data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications from sequencing data. In this study, we propose a bio-computational framework, Nm-Nano, for predicting the presence of Nm sites in direct RNA sequencing data generated from two human cell lines. The Nm-Nano framework integrates two supervised machine learning (ML) models for predicting Nm sites: Extreme Gradient Boosting (XGBoost) and Random Forest (RF) with K-mer embedding. Evaluation on benchmark datasets from direct RNA sequecing of HeLa and HEK293 cell lines, demonstrates high accuracy (99% with XGBoost and 92% with RF) in identifying Nm sites. Deploying Nm-Nano on HeLa and HEK293 cell lines reveals genes that are frequently modified with Nm. In HeLa cell lines, 125 genes are identified as frequently Nm-modified, showing enrichment in 30 ontologies related to immune response and cellular processes. In HEK293 cell lines, 61 genes are identified as frequently Nm-modified, with enrichment in processes like glycolysis and protein localization. These findings underscore the diverse regulatory roles of Nm modifications in metabolic pathways, protein degradation, and cellular processes. The source code of Nm-Nano can be freely accessed at https://github.com/Janga-Lab/Nm-Nano.

Epitranscriptome insights into Riccia fluitans L. (Marchantiophyta) aquatic transition using nanopore direct RNA sequencing.

BACKGROUND: Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergone in response to land-water transition. RESULTS: A significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant's resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions. CONCLUSIONS: These insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution. These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant's dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants.

Transfer learning enables identification of multiple types of RNA modifications using nanopore direct RNA sequencing.

Nanopore direct RNA sequencing (DRS) has emerged as a powerful tool for RNA modification identification. However, concurrently detecting multiple types of modifications in a single DRS sample remains a challenge. Here, we develop TandemMod, a transferable deep learning framework capable of detecting multiple types of RNA modifications in single DRS data. To train high-performance TandemMod models, we generate in vitro epitranscriptome datasets from cDNA libraries, containing thousands of transcripts labeled with various types of RNA modifications. We validate the performance of TandemMod on both in vitro transcripts and in vivo human cell lines, confirming its high accuracy for profiling m6A and m5C modification sites. Furthermore, we perform transfer learning for identifying other modifications such as m7G, Ψ, and inosine, significantly reducing training data size and running time without compromising performance. Finally, we apply TandemMod to identify 3 types of RNA modifications in rice grown in different environments, demonstrating its applicability across species and conditions. In summary, we provide a resource with ground-truth labels that can serve as benchmark datasets for nanopore-based modification identification methods, and TandemMod for identifying diverse RNA modifications using a single DRS sample.

Nanopore sequencing of influenza A and B in Oxfordshire and the United Kingdom, 2022-23.

OBJECTIVES: We evaluated Nanopore sequencing for influenza surveillance. METHODS: Influenza A and B PCR-positive samples from hospital patients in Oxfordshire, UK, and a UK-wide population survey from winter 2022-23 underwent Nanopore sequencing following targeted rt-PCR amplification. RESULTS: From 941 infections, successful sequencing was achieved in 292/388 (75 %) available Oxfordshire samples: 231 (79 %) A/H3N2, 53 (18 %) A/H1N1, and 8 (3 %) B/Victoria and in 53/113 (47 %) UK-wide samples. Sequencing was more successful at lower Ct values. Most same-sample replicate sequences had identical haemagglutinin segments (124/141, 88 %); 36/39 (92 %) Illumina vs. Nanopore comparisons were identical, and 3 (8 %) differed by 1 variant. Comparison of Oxfordshire and UK-wide sequences showed frequent inter-regional transmission. Infections were closely-related to 2022-23 vaccine strains. Only one sample had a neuraminidase inhibitor resistance mutation. 849/941 (90 %) Oxfordshire infections were community-acquired. 63/88 (72 %) potentially healthcare-associated cases shared a hospital ward with ≥ 1 known infectious case. 33 epidemiologically-plausible transmission links had sequencing data for both source and recipient: 8 were within ≤ 5 SNPs, of these, 5 (63 %) involved potential sources that were also hospital-acquired. CONCLUSIONS: Nanopore influenza sequencing was reproducible and antiviral resistance rare. Inter-regional transmission was common; most infections were genomically similar. Hospital-acquired infections are likely an important source of nosocomial transmission and should be prioritised for infection prevention and control.

Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing.

Whole-genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long- and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long-read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40×depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting.

Direct Analysis of HIV mRNA m6A Methylation by Nanopore Sequencing.

The post-transcriptional processing and chemical modification of HIV RNA are understudied aspects of HIV virology, primarily due to the limited ability to accurately map and quantify RNA modifications. Modification-specific antibodies or modification-sensitive endonucleases coupled with short-read RNA sequencing technologies have allowed for low-resolution or limited mapping of important regulatory modifications of HIV RNA such as N6-methyladenosine (m6A). However, a high-resolution map of where these sites occur on HIV transcripts is needed for detailed mechanistic understanding. This has recently become possible with new sequencing technologies. Here, we describe the direct RNA sequencing of HIV transcripts using an Oxford Nanopore Technologies sequencer and the use of this technique to map m6A at near single nucleotide resolution. This technology also provides the ability to identify splice variants with long RNA reads and thus, can provide high-resolution RNA modification maps that distinguish between overlapping splice variants. The protocols outlined here for m6A also provide a powerful paradigm for studying any other RNA modifications that can be detected on the nanopore platform.

Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow. METHODS: We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case-control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES). RESULTS: Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES. CONCLUSIONS: Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.

Rapid identification of SARS-CoV-2 strains via isothermal enzymatic recombinase amplification and nanopore sequencing.

Surveillance of the SARS-CoV-2 genome has become a crucial technique in the management of COVID-19, aiding the pandemic response and supporting effective public health interventions. Typically, whole-genomic sequencing is used along with PCR-based target enrichment techniques to identify SARS-CoV-2 variants, which is a complicated and time-consuming process that requires central laboratory facilities. Thus, there is an urgent need to develop rapid and cost-effective tools for precise on-site detection and identification of SARS-CoV-2 strains. In this study, we demonstrate the rapid diagnosis of COVID-19 and identification of SARS-CoV-2 variants by amplification and sequencing of the entire SARS-CoV-2 S gene using isothermal enzymatic recombinase amplification combined with the advanced Oxford nanopore sequencing technique. The entire procedure, from sampling to sequencing, takes less than 8 hours and can be performed with limited resources. The newly developed method has noteworthy implications for examining the transmission dynamics of the virus, detecting novel genetic variants, and assessing the effect of mutations on diagnostic approaches, antiviral treatments, and vaccines.

Streamlining remote nanopore data access with slow5curl.

BACKGROUND: As adoption of nanopore sequencing technology continues to advance, the need to maintain large volumes of raw current signal data for reanalysis with updated algorithms is a growing challenge. Here we introduce slow5curl, a software package designed to streamline nanopore data sharing, accessibility, and reanalysis. RESULTS: Slow5curl allows a user to fetch a specified read or group of reads from a raw nanopore dataset stored on a remote server, such as a public data repository, without downloading the entire file. Slow5curl uses an index to quickly fetch specific reads from a large dataset in SLOW5/BLOW5 format and highly parallelized data access requests to maximize download speeds. Using all public nanopore data from the Human Pangenome Reference Consortium (>22 TB), we demonstrate how slow5curl can be used to quickly fetch and reanalyze raw signal reads corresponding to a set of target genes from each individual in large cohort dataset (n = 91), minimizing the time, egress costs, and local storage requirements for their reanalysis. CONCLUSIONS: We provide slow5curl as a free, open-source package that will reduce frictions in data sharing for the nanopore community: https://github.com/BonsonW/slow5curl.

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.

Comprehensive identification of pathogenic microbes and antimicrobial resistance genes in food products using nanopore sequencing-based metagenomics.

Foodborne pathogens, particularly antimicrobial-resistant (AMR) bacteria, remain a significant threat to global health. Given the limitations of conventional culture-based approaches, which are limited in scope and time-consuming, metagenomic sequencing of food products emerges as a promising solution. This method provides a fast and comprehensive way to detect the presence of pathogenic microbes and antimicrobial resistance genes (ARGs). Notably, nanopore long-read sequencing provides more accurate bacterial taxonomic classification in comparison to short-read sequencing. Here, we revealed the impact of food types and attributes (origin, retail place, and food processing methods) on microbial communities and the AMR profile using nanopore metagenomic sequencing. We analyzed a total of 260 food products, including raw meat, sashimi, and ready-to-eat (RTE) vegetables. Clostridium botulinum, Acinetobacter baumannii, and Vibrio parahaemolyticus were identified as the top three foodborne pathogens in raw meat and sashimi. Importantly, even with low pathogen abundance, higher percentages of samples containing carbapenem and cephalosporin resistance genes were identified in chicken and RTE vegetables, respectively. In parallel, our results demonstrated that fresh, peeled, and minced foods exhibited higher levels of pathogenic bacteria. In conclusion, this comprehensive study offers invaluable data that can contribute to food safety assessments and serve as a basis for quality indicators.

Nanopore and Illumina sequencing reveal different viral populations from human gut samples.

The advent of viral metagenomics, or viromics, has improved our knowledge and understanding of global viral diversity. High-throughput sequencing technologies enable explorations of the ecological roles, contributions to host metabolism, and the influence of viruses in various environments, including the human intestinal microbiome. However, bacterial metagenomic studies frequently have the advantage. The adoption of advanced technologies like long-read sequencing has the potential to be transformative in refining viromics and metagenomics. Here, we examined the effectiveness of long-read and hybrid sequencing by comparing Illumina short-read and Oxford Nanopore Technology (ONT) long-read sequencing technologies and different assembly strategies on recovering viral genomes from human faecal samples. Our findings showed that if a single sequencing technology is to be chosen for virome analysis, Illumina is preferable due to its superior ability to recover fully resolved viral genomes and minimise erroneous genomes. While ONT assemblies were effective in recovering viral diversity, the challenges related to input requirements and the necessity for amplification made it less ideal as a standalone solution. However, using a combined, hybrid approach enabled a more authentic representation of viral diversity to be obtained within samples.

High-resolution and real-time wastewater viral surveillance by Nanopore sequencing.

Wastewater genomic sequencing stands as a pivotal complementary tool for viral surveillance in populations. While long-read Nanopore sequencing is a promising platform to provide real-time genomic data, concerns over the sequencing accuracy of the earlier Nanopore versions have somewhat restrained its widespread application in wastewater analysis. Here, we evaluate the latest improved version of Nanopore sequencing (R10.4.1), using SARS-CoV-2 as the model infectious virus, to demonstrate its effectiveness in wastewater viral monitoring. By comparing amplicon lengths of 400 bp and 1200 bp, we revealed that shorter PCR amplification is more suitable for wastewater samples due to viral genome fragmentation. Utilizing mock wastewater samples, we validated the reliability of Nanopore sequencing for variant identification by comparing it with Illumina sequencing results. The strength of Nanopore sequencing in generating real-time genomic data for providing early warning signals was also showcased, indicating that as little as 0.001 Gb of data can provide accurate results for variant prevalence. Our evaluation also identified optimal alteration frequency cutoffs (>50 %) for precise mutation profiling, achieving >99 % precision in detecting single nucleotide variants (SNVs) and insertions/deletions (indels). Monitoring two major wastewater treatment plants in Hong Kong from September 2022 to April 2023, covering over 4.5 million population, we observed a transition in dominant variants from BA.5 to XBB lineages, with XBB.1.5 being the most prevalent variants. Mutation detection also highlighted the potential of wastewater Nanopore sequencing in uncovering novel mutations and revealed links between signature mutations and specific variants. This study not only reveals the environmental implications of Nanopore sequencing in SARS-CoV-2 surveillance but also underscores its potential in broader applications including environmental health monitoring of other epidemic viruses, which could significantly enhance the field of wastewater-based epidemiology.

An evolution of Nanopore next-generation sequencing technology: implications for medical microbiology and public health.

Next-generation sequencing has evolved as a powerful tool, with applications that extend from diagnosis to public health surveillance and outbreak investigations. Short-read sequencing, using primarily Illumina chemistry, has been the prevailing approach. Single-molecule sensing and long-read sequencing using Oxford Nanopore Technologies (ONT) has witnessed a breakthrough in the evolution of the technology, performance, and applications in the past few years. In this issue of the Journal of Clinical Microbiology, Bogaerts et al. (https://doi.org/10.1128/jcm.01576-23) describe the utility of the latest ONT sequencing technology, the R10.4.1, in bacterial outbreak investigations. The authors demonstrate that ONT R10.4.1 technology can be comparable to Illumina sequencing for single-nucleotide polymorphism-based phylogeny. The authors emphasize that the reproducibility between ONT and Illumina technologies could facilitate collaborations among laboratories utilizing different sequencing platforms for outbreak investigations.