Discovery of a Novel HLA-G Allele, HLA-G*01:64, by PolyseqOne and Oxford Nanopore Sequencing.
HLA-G*01:64 differs from HLA-G*01:04:01 by one nonsynonymous nucleotide substitution in codon 107 in exon 2.
Humans↗
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Results for “nanopore sequencing”
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ECHO: a nanopore sequencing-based workflow for (epi)genetic profiling of the human repeatome.
SUMMARY: The human genome is dominated by repetitive DNA, whose genetic and epigenetic variation plays a key role in gene regulation, genome stability, and disease. Recent advances in long-read sequencing now enable large-scale, haplotype-resolved, and DNA methylation-informative analysis of the human genome, including on previously inaccessible complex and repetitive regions. However, the comprehensive, simultaneous characterisation of the "human repeatome" remains challenging, largely due to the lack of comprehensive tools integrated in a single pipeline that can capture the full spectrum of variation across diverse types of DNA repeats. Here, we present ECHO, a user-friendly, Snakemake-based pipeline for the "(Epi)genomic Characterisation of Human Repetitive Elements using Oxford Nanopore Sequencing." ECHO provides a reproducible and scalable framework for end-to-end analysis of whole-genome nanopore sequencing data, enabling integrative but also tailored (epi)genetic analyses of the human repeatome. AVAILABILITY AND IMPLEMENTATION: ECHO is freely available at Github: https://github.com/leenput/ECHO-pipeline, with the archived version at Zenodo: https://zenodo.org/records/19068468.
Humans↗
Parallel Analysis of Repeat Expansions: An Updated Clinical Nanopore Cas9-Targeted Sequencing Workflow for Nanopore R10 Flow Cells.
Hereditary ataxias, caused by expansions of short tandem repeats, are difficult to diagnose using traditional PCR and Southern blot methods, which struggle to detect complex repeat expansions and cannot assess repeat interruptions or methylation. An updated Clinical Nanopore Cas9-Targeted Sequencing workflow is presented for analyzing repeat expansions, now compatible with the Oxford Nanopore Technologies R10 flow cell. The workflow incorporates the Oxford Nanopore Technologies wf-human-variation Epi2Me workflow, including the Straglr tool to analyze base-called reads, ensuring compatibility with past, current, and future sequencing chemistries. It expands the number of genes analyzed from 10 to 27 and introduces new gene panels for ataxia, myopathy, neurodegeneration, and amyotrophic lateral sclerosis/motor neuron disease. Validated with Coriell reference and clinical samples, this method improves the analysis of pathogenic repeat expansions, providing deeper insights into repeat structures while addressing the limitations of traditional approaches. In this work, the use of multiplexing, Flongle flow cells, and single-gene targeting were explored as alternatives to panel-based approaches in the Clinical Nanopore Cas9-Targeted Sequencing workflow, finding that only single-gene targeting provides compatibility and reliable performance.
Journal Article↗
Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.
Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.
RNA↗
Nanopore-based sequencing of active DNA replication reveals key principles of metazoan replication fork progression, origin and termination sites.
Balancing replication fork progression and origin usage is essential to maintain genome stability, but measuring replication fork progression rates and origin usage throughout the genome has been challenging. Here, we use nanopore sequencing combined with DNAscent to measure replication fork progression together with origin and termination site usage with single-molecule precision throughout the Drosophila genome with nearly full genome coverage. We find that replication fork progression rates are not uniform throughout the genome. Rather, fork progression is slowest in euchromatin, and this is not correlated with active transcription. Replication origins are also influenced by chromatin, but the exact position of initiation is highly variable and are often several kilobases away from ORC binding sites. Termination sites lack any chromatin or sequence motifs and appear nearly random throughout the genome. By measuring DNA replication dynamics at near full genome coverage, our work reveals key principles of metazoan replication dynamics.
Journal Article↗
Optimized Amplicon Strategy for Long-Read Sequencing of the Chikungunya Virus Genome.
Chikungunya virus (CHIKV) is a positive-sense RNA alphavirus transmitted to humans primarily by Aedes aegypti and Aedes albopictus mosquitoes. Its global circulation and significant public health impact underscore the need to better understand the molecular mechanisms driving CHIKV pathogenesis and transmission. Although robust molecular biology methods exist for CHIKV genome sequencing, a major limitation for surveillance and research is the inability to determine whether two nucleotide variations co-occur within the same viral genome when they are separated beyond the span of typical short-read designs. Here, we describe an optimized approach for processing CHIKV RNA samples that generates large amplicons suitable for long-read nanopore sequencing. This protocol enables amplification of the complete CHIKV genome in only two or three amplicons and facilitates detection of co-occurring nucleotide variations across 4-7.5 kb within the same molecule, thereby simplifying sequencing workflows and improving resolution in studies of viral evolution.
Chikungunya virus↗
A parallel graph decomposition algorithm for DNA sequencing with nanopores.
MOTIVATION: With the potential availability of nanopore devices that can sense the bases of translocating single-stranded DNA (ssDNA), it is likely that 'reads' of length approximately 10(5) will be available in large numbers and at high speed. We address the problem of complete DNA sequencing using such reads. We assume that approximately 10(2) copies of a DNA sequence are split into single strands that break into randomly sized pieces as they translocate the nanopore in arbitrary orientations. The nanopore senses and reports each individual base that passes through, but all information about orientation and complementarity of the ssDNA subsequences is lost. Random errors (both biological and transduction) in the reads create further complications. RESULTS: We have developed an algorithm that addresses these issues. It can be considered an extreme variation of the well-known Eulerian path approach. It searches over a space of de Bruijn graphs until it finds one in which (a) the impact of errors is eliminated and (b) both possible orientations of the two ssDNA sequences can be identified separately and unambiguously. Our algorithm is able to correctly reconstruct real DNA sequences of the order of 10(6) bases (e.g. the bacterium Mycoplasma pneumoniae) from simulated erroneous reads on a modest workstation in about 1 h. We describe, and give measured timings of, a parallel implementation of this algorithm on the Cray Multithreaded Architecture (MTA-2) supercomputer, whose architecture is ideally suited to this 'unstructured' problem. Our parallel implementation is crucial to the problem of rapidly sequencing long DNA sequences and also to the situation where multiple nanopores are used to obtain a high-bandwidth stream of reads.
Algorithms↗
Recognizing a single base in an individual DNA strand: a step toward DNA sequencing in nanopores.
Explore the source record for details and available documents.
Bacterial Toxins↗
NanoASV: a snakemake workflow for reproducible field-based Nanopore full-length 16S metabarcoding amplicon data analysis.
SUMMARY: NanoASV is a conda environment and snakemake-based workflow using state-of-the-art bioinformatics software to process full-length SSU rRNA (16S/18S) amplicons acquired with Oxford Nanopore Sequencing technology. Its strength lies in reproducibility, portability, and the possibility to run offline, allowing in-field analysis. It can be installed on the Nanopore MK1C sequencing device and process data locally. AVAILABILITY AND IMPLEMENTATION: Source code and documentation are freely available at https://github.com/ImagoXV/NanoASV and Zenodo archive at https://doi.org/10.5281/zenodo.14730742.
Software↗
Cross-Platform Methylation-Based Site of Origin Classification for Squamous Cell Carcinomas.
Squamous cell carcinomas (SCCs) are one of the most common cancer types and can arise at nearly any anatomic site. Because SCCs are one of the most common metastases, do not have reliable site-specific morphologic or genomic features, and have considerable morphologic and immunohistochemical overlap with urothelial carcinomas, distinguishing between primary and metastatic squamous-appearing tumors can be challenging. This distinction can be critical to clinical management. We present Squamous cell carcinoma Methylation for Origin Site (SquaMOS), a methylation-based classifier to predict site of origin of squamous-appearing carcinomas. Trained on publicly available array-based methylation data from 1062 primary SCCs (from lung, head and neck, cervix, and esophagus) and urothelial carcinomas, SquaMOS predicted site of origin in primary tumors with 96.1% accuracy in an internal test set (n = 458) and 97.4% accuracy in an external test set from 3 institutions (n = 78). On metastatic tumors (n = 51), SquaMOS predictions were 96.1% accurate. SquaMOS was directly applicable to shallow Nanopore sequencing data (CpG probe site coverage, 0.25-2.88×) with an accuracy of 91.7% (n = 36; 100% accurate for high-confidence predictions). When tested on SCCs outside the training set types (n = 15, including 3 metastases to lung), no cases were misclassified as of lung origin, supporting accuracy of lung vs nonlung origin classification for diverse SCC types. Overall, we demonstrate highly accurate performance of the SquaMOS classifier on primary and metastatic tumors from multiple data sources, robust to suboptimal tumor purity. We illustrate transferability of our array-based classifier to low-depth Nanopore sequencing data, a potentially rapid means of site of origin determination in a clinical setting.
Humans↗
VisPan: real-time visualisation of multiplex amplicon-based sequencing panels for rapid syndromic surveillance and pathogen detection.
MOTIVATION: Infectious diseases persist as a major global public health challenge. Diverse factors, including climate change, globalization, deforestation, human-animal interactions, lifestyle choices, and various biological factors, can contribute to their emergence and reemergence. Rapid detection and characterization of (re)emerging pathogens are therefore critical for effective outbreak management and for enhancing our understanding of epidemics by monitoring the transmission, spread, evolution, and genomics of pathogens. In this context, next-generation sequencing technologies (NGS), particularly long-read platforms such as Oxford Nanopore Technologies (ONT), have opened new avenues for real-time pathogen monitoring. However, the bioinformatics bottleneck remains a challenge, emphasizing the need for efficient, accessible, and user-friendly analysis tools. RESULTS: Here, we present a tool adapted from the RAMPART software that enables real-time data visualisation of multiplex PCR syndromic panels combined with Oxford Nanopore sequencing. This real-time analysis enables rapid pathogen detection, from raw data acquisition to taxonomic assignment, within minutes. The interface offers dynamic visual tracking of the sequencing run and amplicon coverage, facilitating immediate insights during diagnostic workflows. Validation experiments confirmed the system's reliability, accurately identifying all pathogens present in complex clinical or environmental samples. This tool provides an integrated, user-friendly solution for genomic pathogen surveillance in field or clinical settings.
Software↗
High-quality chromosome-level genome of three Meretrix species using Nanopore and Hi-C technologies.
Meretrix is a commercially valuable bivalve genus in Asia, but only one reference genome has hindered comprehensive genetic studies and germplasm resource evaluation. In this study, we present three reference genomes of Meretrix species: Meretrix sp. MF1, Meretrix sp. MT1, and Meretrix lamarckii JML1. Meretrix sp. MF1 was assembled at the chromosome level using Nanopore sequencing and Hi-C technologies, whereas Meretrix sp. MT1 and Meretrix lamarckii were assembled as scaffold-level assemblies. The chromosome-level genome of Meretrix sp. MF1 consists of 36 contigs, including 19 chromosomes and 17 scaffolds, with a total length of 883.3 Mb and a scaffold N50 of 46.87 Mb. Notably, the genome of Meretrix sp. MF1, a putative novel species, exhibits an Average Nucleotide Identity (ANI) of 94.33% with its closest relative, Meretrix lamarckii. These genomic resources not only provide a crucial foundation for genetic research on Meretrix but also contribute to the development of effective conservation strategies for its sustainable management.
Animals↗
Long-Read Haplotype Phasing Resolves Allelic Configuration as a Missing Layer of Precision Oncology.
Short-read sequencing cannot determine whether co-occurring variants within a cancer gene lie on the same allele (cis) or opposing alleles (trans), a distinction with direct therapeutic consequences: trans configurations confirm biallelic tumor suppressor inactivation, whereas cis configurations generate compound oncogenic alleles with enhanced activity. Among 768 patients with prostate, breast, or ovarian cancers, we used mutational signatures to nominate cryptic genomic instability cases lacking a causative biallelic event on short-read sequencing. Long-read nanopore sequencing resolved 32 of 46 cryptic cases (69.6%) through methylation detection, long insertion resolution, and structural variant characterization, confirming trans inactivation in every resolved tumor suppressor case. Analysis of 4,496 MiOncoSeq samples identified 17,519 multi-hit gene pairs, 78.7% of which exceeded the 500 bp short-read phasing limit, and long-read phasing revealed recurrent compound cis alleles in NOTCH1, PIK3CA, PDGFRB, and KIT. Haplotype phasing addresses an overlooked gap in cancer variant interpretation and warrants integration into precision oncology.
Journal Article↗
SMART-RNA-Metavirome: a practical RNA metavirome platform compatible with high-throughput sequencing of both short and long reads.
BACKGROUND: The RNA virosphere's extensive diversity and its role in emerging infectious diseases underscore the importance of non-targeted sequencing for identifying unknown or rare pathogens, including co-infections. However, enriching low-abundance viral sequences in RNA metaviromics, particularly in the preparation of cDNA libraries and their compatibility with next-generation sequencing (NGS) and third-generation sequencing (TGS), remains challenging. Therefore, our objective is to develop and systematically assess a practical RNA metavirome methodology specifically tailored for the enrichment of low-abundance viral sequences within samples. METHODS: We developed the SMART-RNA-Metavirome platform, integrating SMART-9n library preparation with NGS and TGS technologies. Total RNA was extracted from two field-collected wild Aedes albopictus pools, along with one laboratory-infected Ae. albopictus pool harboring dengue virus (DENV). This RNA was subjected to reverse transcription using both this optimized protocol and random primer-based methods, followed by high-throughput sequencing on Illumina, Oxford Nanopore, and QitanTech Nanopore technologies. Welch's t-test was employed for comparative analysis of the subsequent RNA metavirome data, specifically to evaluate differences in viral species composition and abundance of viral reads between experimental groups. Furthermore, the effectiveness of this platform was systematically validated via RT-qPCR and SMART-RNA-Metavirome-based Oxford Nanopore sequencing across multiple sample types, including mosquito specimens from DENV-infected Ae. albopictus, serum samples from dengue patients and viral isolates of Japanese encephalitis virus (JEV) and Zika virus (ZIKV). RESULTS: The SMART-RNA-Metavirome platform has been systematically validated to excel in enriching the composition and diversity of the RNA virome (P = 0.04), providing sufficient coverage for the complete reconstruction of viral genomes. When employed in the detection of DENV-infected Ae. albopictus, clinical serum samples, and viral isolates of JEV and ZIKV, this technique exhibits a robust correlation with RT-qPCR (r2 > 0.95). Notably, it demonstrates exceptional sensitivity, ensuring sufficient coverage even in samples of DENV-infected Ae. albopictus with a Ct-value of 35.3, attaining an impressive 99.88% genome coverage. Furthermore, this platform possesses the capability to identify virus species and determine their serotypes. CONCLUSIONS: In our study, the SMART-RNA-Metavirome platform outperforms traditional methods, enriching RNA virome composition and diversity, enabling practical compatibility with both NGS and TGS technologies. It demonstrates significant proficiency in detecting both known and unknown arboviruses, even in low-titer samples such as those from wild mosquitoes and clinical sera. This platform facilitates comprehensive monitoring, risk assessment, and early warning of RNA virus transmissions, enhancing our understanding of RNA virome diversity and ecological patterns.
High-Throughput Nucleotide Sequencing↗
Long-Read Haplotype Phasing Resolves Allelic Configuration as a Missing Layer of Precision Oncology.
Conventional short-read sequencing cannot determine whether co-occurring variants within a cancer gene reside on the same allele (cis) or on opposing alleles (trans), a distinction with direct biological and therapeutic consequences. Trans configurations confirm biallelic tumor suppressor inactivation and inform therapy selection, while cis configurations generate compound oncogenic alleles with enhanced activity. We analyzed 768 patients with prostate, breast, or ovarian cancers in the PROBLEM cohort, using mutational signatures to nominate cryptic genomic instability cases where the causative biallelic event was not apparent from short-read sequencing. Long-read nanopore sequencing resolved 32 of 46 cryptic cases (69.6%), leveraging its unique advantages in direct methylation detection, long insertion resolution, and complex structural variant characterization, confirming trans biallelic inactivation in all resolved tumor suppressor cases. Systematic analysis of 4,496 MiOncoSeq samples identified 17,519 multi-hit gene pairs, of which 78.7% exceeded the 500 bp short-read phasing limit. Long-read phasing further revealed recurrent compound cis oncogenic alleles in NOTCH1, PIK3CA, PDGFRB, and KIT with functionally synergistic activity. Haplotype phasing resolves a systematically overlooked gap in cancer variant interpretation and warrants broader integration into precision oncology workflows.
Journal Article↗
Allele Level Sequencing of Killer Cell Immunoglobulin-Like Receptor Genes Using Oxford Nanopore Long Read Sequencing.
The human Killer cell Immunoglobulin-like Receptor (KIR) genes, found on chromosome 19, encode for cell surface protein receptors that, through interaction with their ligand, modulate the action of Natural Killer (NK) cells and some subsets of T lymphocytes. KIR genes exhibit extensive variation through variable gene content, copy number, and allele polymorphism. The combination of KIR genes and their ligands is implicated in various clinical settings including haematopoietic stem cell and solid organ transplant, and infectious disease progression. KIR gene content has been used in the selection of optimal stem cell donors with haplotype variations in recipient and donor giving differential clinical outcomes. With the introduction of massively parallel clonal next generation sequencing and single molecule long read third generation sequencing, allele level determination of KIR genotypes has become feasible. We describe a method for amplicon-based long read sequencing on the Oxford Nanopore Technologies platform that provides largely unambiguous allele level typing of KIR genes. The method was validated using DNA extracted from 48 10th International Histocompatibility Workshop (IHWS) cell lines with previously published allele level KIR genotypes and 176 Western Australian samples previously tested for the presence or absence of KIR genes. Our long-read sequencing method was able to accurately determine KIR alleles with an overall concordance of 97%-99% with the published data. Importantly, phasing ambiguity caused by the inability to phase heterozygous base positions over long stretches of gene sequence was resolved in several samples. Thus, our long read PCR sequencing strategy can be used to determine KIR genotypes at allele resolution level.
Humans↗
Accurate serotype identification of Streptococcus pneumoniae using nanopore Cas9-targeted serotype identification (nCATSerotyping).
Streptococcus pneumoniae (pneumococcus) is a leading cause of community-acquired pneumonia and invasive diseases, particularly among children and the elderly. The introduction of pneumococcal conjugate vaccines has significantly reduced invasive pneumococcal disease, but the prevalence of non-vaccine serotypes and newly emerging serotypes is increasing globally. Thus, accurate serotyping is essential for epidemiological surveillance and the development of next-generation multivalent pneumococcal vaccines. Conventional serotyping methods, including multiplex polymerase chain reaction (mPCR), monoclonal antibody (mAb) assays, and Quellung reaction using rabbit antisera, are limited by serotype coverage and cross-reactivity, making the detection of new or emerging serotypes challenging. In this study, we developed a nanopore Cas9-targeted serotyping (nCATSerotyping) platform, which employs Cas9-mediated enrichment of the capsular polysaccharide synthesis locus followed by Oxford Nanopore sequencing. Applying this method to 276 clinical pneumococcal isolates collected in South Korea (2018-2020), we achieved a serotyping success rate of 97.10% (268/276), significantly outperforming conventional methods such as mAb and mPCR, which identified only 76.45% (211/276) of isolates. Whole-genome sequencing of the remaining eight non-typeable isolates revealed them to be non-pneumococcal (oral streptococci), confirming 100% accuracy for S. pneumoniae serotyping. Importantly, our method identified emerging and underrepresented serotypes, including serotype 13 and null capsule clade strains. nCATSerotyping offers a rapid, accurate, and comprehensive solution for pneumococcal serotyping, with significant advantages in identifying novel and non-typeable strains. This scalable platform will be a valuable tool for global serotype surveillance and next-generation multivalent pneumococcal vaccine development.IMPORTANCEAccurate pneumococcal serotyping is critical for vaccine development and epidemiological surveillance, particularly as non-vaccine serotypes emerge following widespread pneumococcal conjugate vaccine implementation. Current serotyping methods face significant limitations in coverage and accuracy, identifying around 76% of pneumococcal isolates and failing to detect emerging serotypes like serotype 13 and null capsule clades. The nanopore Cas9-targeted serotyping platform addresses these critical gaps by achieving 100% serotyping accuracy for confirmed Streptococcus pneumoniae isolates while identifying previously undetectable strains that conventional methods missed. This comprehensive approach is essential for monitoring vaccine effectiveness, understanding serotype replacement patterns, and informing next-generation vaccine development strategies. Furthermore, the identification of misclassified oral streptococci highlights the diagnostic precision needed for accurate pneumococcal surveillance, ensuring that epidemiological data accurately reflect true pneumococcal disease burden and serotype distribution patterns.
Streptococcus pneumoniae↗
Emerging protein sequencing technologies: proteomics without mass spectrometry?
INTRODUCTION: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been a leading method for proteomics for 30 years. Advantages provided by LC-MS/MS are offset by significant disadvantages, including cost. Recently, several non-mass spectrometric methods have emerged, but little information is available about their capacity to analyze the complex mixtures routine for mass spectrometry. AREAS COVERED: We review recent non-mass-spectrometric methods for sequencing proteins and peptides, including those using nanopores, sequencing by degradation, reverse translation, and short-epitope mapping, with comments on bioinformatics challenges, fundamental limitations, and areas where new technologies will be more or less competitive with LC-MS/MS. In addition to conventional literature searches, instrument vendor websites, patents, webinars, and preprints were also consulted to give a more up-to-date picture. EXPERT OPINION: Many new technologies are promising. However, demonstrations that they outperform mass spectrometry in terms of peptides and proteins identified have not yet been published, and astute observers note important disadvantages, especially relating to the dynamic range of single-molecule measurements of complex mixtures. Still, even if the performance of emerging methods proves inferior to LC-MS/MS, their low cost could create a different kind of revolution: a dramatic increase in the number of biology laboratories engaging in new forms of proteomics research.
Proteomics↗