Identification of the Novel HLA-E*01:154 Allele Using PolyseqOne and Oxford Nanopore Sequencing Technology.
HLA-E*01:154 differs from HLA-E*01:01:01:01 by a single nonsynonymous nucleotide substitution in codon 199 of exon 3.
Humans↗
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Results for “nanopore sequencing technology”
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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↗
Molecular Epidemiology of Human Metapneumovirus in Kilifi, Coastal Kenya, 2016-2017 and 2021-2024.
BACKGROUND: Human metapneumovirus (hMPV) is a major contributor of acute respiratory infections (ARI) in childhood and vulnerable adults. It comprises two antigenically distinct lineages (A and B), with multiple sub-lineages. Genomic analyses of hMPV strains enable monitoring of viral evolution and transmission to inform future interventions but remain underutilized in Africa. METHODS: We generated 52 near-complete hMPV genomes from respiratory samples collected in Kilifi, Coastal Kenya, using a tiled-amplicon approach and Oxford Nanopore Technologies sequencing. These samples had been identified as hMPV positive by quantitative PCR during (a) a multi-facility outpatient ARI surveillance in nine health facilities in Kilifi between 2016 and 2017, and 2021 to 2023 and (b) a community-based respiratory infection cohort surveillance study between 2023-2024 that sampled enrolled participants irrespective of symptom status. RESULTS: Of the 192 positive samples analyzed from the two studies, children under 5 years accounted for most hMPV cases (134/186, 72%). 52 samples were sequenced (>70% genome coverage), and hMPV-A (27/52, 53.8%) and hMPV-B (25/52, 46.2%) lineages were identified. The recovered sequences mapped into sub-lineages A2c (27/52, 53.8%), B1 (12/52, 21.2%), and B2b (13/52, 25%). A shift in the predominant sub-lineage was observed from B2b (2016) to B1 (2021), and finally to A2c-wild type (2023). In February 2021, for the first time, we detected a single A2c strain with a 111-nucleotide duplication in the G gene among Kenyan samples. CONCLUSION: Our study expands the global nucleotide sequence database for hMPV by adding new whole-genome sequences from Kenya collected over the last decade. It highlights the ongoing replacement of locally predominant hMPV lineages and the importation and local transmission of globally circulating strains. These findings underscore the importance of sustained hMPV genomic surveillance to detect emerging variants and monitor lineage circulation patterns that may impact viral transmission, molecular detection, and future control measures.
A2c-111nt-dup↗
A de novo algorithm for allele reconstruction from Oxford nanopore amplicon reads, with application to CYP2D6.
MOTIVATION: The Oxford Nanopore Technologies' sequencing platform offers a path towards bedside genomics, producing long reads that can completely cover a gene of interest, and detect any known or novel variant the gene contains. However, the analysis of these long reads to identify actionable genotypes remains challenging and typically requires customization depending on the target gene. RESULTS: Here, we describe a generic algorithm to accurately reconstruct allele sequences derived from long-reads of amplicon-based data. Rather than calling variants directly from these long-reads, our method takes a "sequence-first" approach, performing an unbiased reconstruction of the underlying amplicon sequences to generate high-confidence reconstructed allele sequences. This is done without user input of the target gene, allowing for any source amplicon to be reconstructed. These high-confidence reconstructed allele sequences are then compared to the genomic reference sequence of the gene to infer the specific diplotype present in the sample. This approach is agnostic towards the number of genes and alleles present and readily detects novel variants. We demonstrate our approach using three independent data sets for CYP2D6, a diverse and complex gene with over 175 known alleles of clinical significance. We show how our approach can accurately recover validated CYP2D6 diplotypes from 20 Coriell samples covering 14 distinct alleles, using different amplicons, flow cell versions, and depths. This includes inferring occurrences of allele duplication events from relative abundances of each allele, a critical factor for ascribing functional effects to a diplotype. Further, we demonstrate our approach's utility for other genomic regions, including HLA. AVAILABILITY: Custom code is available at the following GitHub repository, along with instructions for use and test data: https://github.com/scottdbrown/allele-reconstruction-long-read-amplicon-data. A snapshot of the code at the time of publication is available on Zenodo.org; doi 10.5281/zenodo.19716004. Raw .fastq sequence data for our three sequencing runs is available at the SRA under Bioproject PRJNA1357883 (https://www.ncbi.nlm.nih.gov/bioproject/1357883).
Alleles↗
Epigenetic Liquid Biopsy Enables Universal Mutation-Agnostic Molecular Surveillance for High-Risk Neuroblastoma.
PURPOSE: Liquid biopsy monitoring in pediatric solid tumors is limited by low mutational burden and lack of trackable genomic drivers. We sought to develop a mutation-agnostic, methylation-based liquid biopsy framework enabling universal molecular surveillance of high-risk neuroblastoma. EXPERIMENTAL DESIGN: Using whole-genome Oxford Nanopore Technologies sequencing of high-risk neuroblastoma tumors, we compared tumor-derived methylation profiles with a comprehensive atlas of normal human cell types and identified 72 neuroblastoma-specific differentially methylated regions (meNBL) that were reliably detectable in cell-free DNA (cfDNA). Marker robustness and specificity were validated using independent neuroblastoma methylation datasets and assessed against methylation profiles from other cancer types. We established neuroblastoma as a distinct methylation entity within the reference atlas by integrating a panel of 25 meNBLs, enabling quantitative estimation of tumor-derived cfDNA. Assay performance was evaluated across diagnostic, remission, relapse, and healthy control samples and compared with mutation-based and copy number-based approaches. RESULTS: Neuroblastoma-derived cfDNA was consistently detected at diagnosis and relapse but was absent in healthy controls and during confirmed remission. Methylation-based deconvolution demonstrated high specificity, with no detectable background signal in controls, and improved performance relative to copy number-based tumor fraction estimation. Longitudinal profiling enabled early molecular detection of relapse and reliable disease monitoring. CONCLUSIONS: We establish a robust, mutation-independent methylation-based liquid biopsy strategy for neuroblastoma that enables accurate, quantitative disease monitoring across all high-risk patients, including those lacking trackable genomic alterations. This approach supports the clinical translation of methylation-based cfDNA deconvolution as a broadly applicable platform for pediatric precision oncology.
Humans↗
Assembly and characterization of the first complete mitochondrial genome of Epimedium sagittatum (Sieb. et Zucc.) Maxim (Berberidaceae):an invaluable traditional Chinese medicine.
BACKGROUND: Epimedium sagittatum (Sieb. et Zucc.) Maxim is an invaluable traditional Chinese medicine plant known for its properties of tonifying kidney yang, strengthening bones and muscles, and dispelling rheumatism. The chloroplast (cp) genome of E. sagittatum have been sequenced, offering critical insights for breeding and phylogenetic research. However, the mitochondrial (mt) genome of E. sagittatum remains uncharacterized, limiting comprehensive insights into its genomic evolution. RESULTS: In this study, we assembled the first complete mt genome of E. sagittatum employing Illumina and Nanopore sequencing technology and subsequently investigated comparative analysis with its closely related species. The mt genome of E. sagittatum was assembled as a multi-branched structure with a length of 339,191 bp, within a GC content of 46.91%. Our annotation results have shown 39 protein-coding genes (PCGs), 22 tRNA genes, three rRNA genes and four pseudogenes in the E. sagittatum mt genome. The analysis of sequence repeats has detected 79 simple sequence repeats (SSRs), 10 tandem repeats and 255 dispersed repeats in the E. sagittatum mt genome. A total of 720 C to U RNA editing sites of the 34 PCGs was predicted in E. sagittatum. The codons exhibited a strong preference for A or U bases in the E. sagittatum mt genome. The analysis of nucleotide diversity (Pi) highlighted differences in genetic variability across the tested genes, with atp9 gene exhibiting the highest genetic variation. Selection pressure analysis showed that most genes were affected by negative selection during evolution, whereas ccmB, rps10, and rps12 underwent positive selection in different plants. Additionally, a Bayesian phylogenetic tree showed that E. sagittatum was closely related to E. wushanense and E. pubescens. In total of 14 homologous fragments totaling 8,954 bp were identified between the cp and mt genomes of E. sagittatum. CONCLUSIONS: This study presents the first assembled and annotated mt genome of E. sagittatum, which provides a valuable genetic resource for the Epimedium genus and lays the foundation for investigating the phylogenetic relationship and genetic variation of this invaluable medicinal plant.
Epimedium↗
New insights on Plasmodium gene expression from direct RNA sequencing.
Oxford Nanopore Technology (ONT) direct RNA sequencing enables the sequencing of native RNA molecules without cDNA conversion. The long-read approach captures full-length reads spanning entire genes and has transformed the study of gene expression in Plasmodium parasites by enabling analysis of untranslated regions, isoforms, and alternative splicing. In addition, ONT provides unique insights into non-coding RNAs, RNA modifications, and polyadenylated tail dynamics, which are expanding our understanding of post-transcriptional regulation in Plasmodium, including processes beyond translational repression in gametocytes and sporozoites. Here, we discuss the past and future applications of direct RNA sequencing in Plasmodium research and highlight its advantages, limitations, and future prospects.
Oxford Nanopore Technology↗
OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides.
Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands (e.g., over a million nucleotides long) for genomics research and biotechnology applications. However, the same level of progress has not been achieved for DNA oligonucleotides (usually ≤ 300 nucleotides long). Oligonucleotides play a crucial role in genome engineering efforts through oligo library generation and in DNA data storage, where they are used to encode computer information, such as binary (digital) data in DNA libraries. To enable these applications, accurate sequencing of oligonucleotides in a way that allows to assess for sequence variability, quality and length is essential. But sequencing solutions for oligonucleotides - particularly DNA primers for PCR, oligo DNA libraries used for mutagenesis or cDNA libraries used in gene expression analysis - remain inadequate. To address this gap, OligoSeq is presented as an innovative approach that integrates two complementary techniques: AmpliSeq (based on PCR) and RevSeq (based on reverse complementation with sequence-specific or random primers) to facilitate sequencing of single-stranded oligonucleotides using reference sequence anchor matches of more than ≥ 90% identity spanning from about 70% to 10% with AmpliSeq or RevSeq with random nonamers, respectively, and resolving the final reference sequence based on the most likely candidate from basecall frequencies, regardless of length and double-stranding method. OligoSeq can be integrated with nanopore sequencing technology pipelines and can be used as a reference for other sequencing platforms requiring double-stranded adapters, offering a practical and scalable alternative for standard quality control in single-stranded oligonucleotide synthesis. The use of nanopore technology, compatible with the double-stranding methods showcased, is shown to be the most cost-effective method for resolving original DNA sequences of different length and quality, and to assess its sequence variability, compared to other methods such as Illumina, PacBio or HPLC/MS.
Sequence Analysis, DNA↗
Complete genome of multiply antibiotic resistant ST10 Acinetobacter baumannii isolate NL6 from Vietnam and relationship to available ST10 genomes.
The genome of NL6, a multiply antibiotic-resistant Acinetobacter baumannii ST10:KL49:OCL2 carriage isolate from Vietnam, was sequenced using Nanopore technology, and complete chromosome and plasmid sequences were assembled from the long reads and available short reads. Resistance genes and their locations were identified, and transfer of a conjugative plasmid carrying several resistance genes into a new host was tested. The acquired resistance genes in NL6 were distributed between the chromosome and two of three plasmids present. The chromosome carries multiple copies of several insertion sequences, an incomplete copy of the ISAba1-bounded Tn6250 that includes the sul2 and strAB genes, and an integrative element carrying copper resistance genes designated IECuR. Plasmid pNL6-2 (r3-T5; 15 Kbp) is a Rep_3/OrfX plasmid that includes a tet39 dif module, and pNL6-3 (r3-T20; 66.9 Kbp) carries aacC2d, aphA6, and blaCARB-16 and a second ampC gene preceded by an ISAba1. Conjugation of pNL6-3 into derivatives of ATCC17978 was demonstrated, confirming that the ampC gene confers resistance to third-generation cephalosporins. NL6 was compared to other complete ST10 genomes. Several acquired elements in the chromosome were shared with the ST10 isolate LAC-4 (USA), indicating shared ancestry, but the plasmid content differed. The KL and plasmid content were variable in 17 further complete ST10 genomes downloaded from GenBank. Tn6250 and IECuR were only found together in the chromosome of two further KL49 isolates. Antibiotic resistance in ST10 A. baumannii was acquired mainly via plasmid acquisition, but resistance genes varied, and a variety of plasmids was involved.IMPORTANCEMembers of the CC10 clonal complex of Acinetobacter baumannii comprising ST10 plus single and double locus variants are known to be particularly virulent. However, antibiotic resistance in members of this group has rarely been examined. Here, determination of the complete genome (chromosome and plasmids) of a representative ST10 isolate from Vietnam allowed the context and location of acquired antibiotic resistance genes and of other mobile genetic elements to be determined. Mobile genetic element locations in completed chromosomes facilitate comparisons of potentially related genomes, revealing those with recent shared ancestry. Differences in plasmid content can also be examined.
Acinetobacter baumannii↗
Comparative performance of portable DNA extraction protocols and bioinformatics workflows for rapid detection of gram-negative bacteria and antimicrobial resistance using Oxford Nanopore sequencing.
Oxford Nanopore Technology (ONT) enables rapid, portable pathogen identification and antimicrobial resistance (AMR) detection, but the reliability of downstream genomic analyses is highly dependent on DNA extraction quality, particularly in resource-limited settings. This study comparatively evaluated four portable bacterial DNA extraction protocols derived from three commercial kits to determine their impact on nanopore sequencing performance, bioinformatics workflow completion, and field deployability. Six gram-negative bacterial isolates (Escherichia coli, n = 4; Pseudomonas sp., n = 1; and Salmonella sp., n = 1) were processed using four extraction protocols: SwiftX DNA, SwiftX DNA with proteinase K (ProtK), SwiftX ParaBact, and NucleoSpin Microbial. Twenty-four resulting DNA extracts were sequenced on a single multiplexed MinION R10.4.1 flow cell. Sequencing data were analyzed using validated Galaxy-based generic and species-specific pipelines. Workflow completion was defined as successful progression through quality control, assembly, virulence, plasmid, and AMR detection modules. DNA purity varied substantially by extraction protocol and was strongly associated with successful workflow completion (Kruskal-Wallis, P = 0.0006). Accordingly, NucleoSpin Microbial achieved 100% workflow completion, and SwiftX ParaBact achieved 83%, while both SwiftX DNA-based protocols failed to complete full workflows. Importantly, key AMR genes required to classify isolates as multidrug-resistant were consistently detected using both NucleoSpin Microbial and SwiftX ParaBact extractions. However, NucleoSpin Microbial assemblies showed significantly higher contiguity and enabled a broader, more complete detection of virulence factors, pathogenicity islands, plasmid replicons, and accessory AMR genes, reflecting enhanced genomic resolution.IMPORTANCERapid whole-genome sequencing is increasingly used to detect antimicrobial resistance and guide public health responses, but its reliability depends strongly on how bacterial DNA is extracted. In this study, we have shown that DNA extraction method choice has a major impact on Oxford Nanopore sequencing performance across clinically relevant gram-negative bacteria. While silica column-based extraction maximized genomic completeness and analytical depth, paramagnetic bead-based reverse purification offered superior portability with sufficient resolution for frontline AMR surveillance. These findings highlight a practical trade-off between field deployability and high-resolution genomic characterization in low-resource settings.
DNA extraction↗
Workflow for Long-Read Amplicon Sequencing of Chikungunya Virus Using Oxford Nanopore Technology.
This protocol provides a comprehensive, step-by-step workflow for whole-genome sequencing of Chikungunya virus (CHIKV) using an amplicon-based strategy optimized for Oxford Nanopore Technologies (ONT) platforms. The procedure includes detailed instructions for sample handling, viral RNA extraction, quality control, cDNA synthesis, multiplex PCR amplification, library preparation, sequencing, and primary bioinformatic processing. The protocol is designed to maximize reproducibility across laboratories and is suitable for genomic surveillance applications, including outbreak investigation and molecular epidemiology, even when working with low-to-moderate viral loads.
Chikungunya virus↗
Benchmarking DNA extraction protocols across use cases for culture-independent Nanopore metagenomics.
Oxford Nanopore Technologies (ONT) sequencing offers several advantages for metagenomics, including long reads, rapid turnaround, low upfront cost, scalability and portability. However, for ONT metagenomics, DNA yield, quality and integrity are important considerations when selecting an extraction method. Many metagenomic extraction methods use harsh lysis conditions to extract a wide range of species and provide an accurate community composition, but these conditions can compromise DNA fragment length. Therefore, extraction methods for ONT metagenomics must balance DNA shearing and recovery with representative community lysis. We systematically evaluated DNA extraction methods for ONT metagenomic sequencing using a use case-oriented framework. Among nearly 50 extraction methods screened, 7 were selected for detailed comparison based on suitability for metagenomics, variation in methodology, availability, cost and processing time: Norgen BioTek Corp's Stool DNA Isolation (NG), Zymo Research's ZymoBIOMICS Quick-DNA HMW MagBead (ZMG), Qiagen's DNeasy Blood and Tissue (QBT), Macherey-Nagel's NucleoMag DNA Microbiome (MN), Zymo Research's ZymoBIOMICS DNA Mini Prep (ZMI), Qiagen's DNeasy PowerSoil/QIAamp PowerFecal Pro (PS) and Qiagen's QIAamp Fast DNA Stool Mini (QIA). Methods were tested using Zymo Research's ZymoBIOMICS Microbial Community Standard (MCS), a matrix-free mock community with known composition. DNA extracts were sequenced on an ONT PromethION using the Rapid Barcoding Kit, except QIA due to insufficient DNA yield. Metrics for the method, DNA extracts, sequencing and genomes were evaluated, revealing trade-offs between methods. The two magnetic bead methods, MN and ZMG, produced the highest mean read length N50 values (13.9 and 16.5 kb, respectively) but showed apparent community compositions skewed towards Gram-negative bacteria. In contrast, ZMI and PS maintained a community composition close to expected, with reduced mean read length N50 values (4.5 vs. 7.5 kb). Performance across various metrics is presented in the context of the following use cases: maximizing genome coverage and assembly completeness, preserving composition accuracy, targeting specific species and limiting required resources (equipment, time or budget). The metrics and use case considerations presented offer practical guidance for informed selection of DNA extraction methods for ONT metagenomics. For accurate community composition, ZMI or PS are recommended, while PS and ZMG perform best at maximizing genome coverage and assembly completeness. NG and QBT may be the most economical options, though performance trade-offs were observed. Finally, PS may be the preferred method for time-sensitive diagnostic or field applications.
Metagenomics↗
Leveraging ONT move table values for signal aware variant calling.
Oxford Nanopore Technologies (ONT) sequencing enables long-range haplotype phasing and contiguous genome assembly but still exhibits elevated error rates that challenge small variant calling, particularly for insertions and deletions (Indels). While raw electrical signals contain rich information, existing signal-aware methods require computationally intensive processing of large signal files. Here, we present Clair3 v2, a method that leverages the ONT move table-a lightweight byproduct of basecalling that maps signal events to nucleotide positions-to improve variant calling accuracy. Clair3 v2 builds upon Clair3 and integrates signal-level dwelling time to significantly enhance variant calling performance. We also propose a genome position based circular buffer to incorporate dwelling time with minimal computational overhead. Benchmarking across six Genome in a Bottle samples demonstrates substantial improvements in variant calling accuracy. With HAC basecalling, Clair3 v2 achieves a mean SNP F1-score of 97.69% at 10 × depth (compared to 96.45% for baseline Clair3), and Indel F1 scores improved from 64.27% to 76.70%, while gains persisted at higher depths. The benefits were most pronounced for longer Indels and in complex genomic regions, where Indel F1 scores in long homopolymer regions improved from 14.3% to 45.2%. Benchmark results across various basecalling modes, samples, and coverage settings outperformed Clair3 baselines and other methods, including DeepVariant and Dorado Variant, and demonstrate the significant benefits of Clair3 v2. Furthermore, Clair3 v2 incurs negligible runtime compared to standard Clair3, making it practical for routine use.
Sequence Analysis, DNA↗
Characterisation of the Novel HLA-B*48:43:02 Allele in a Chinese Individual.
HLA-B*48:43:02 differs from HLA-B*48:43:01 by one single nucleotide substitution at position 900 G>C in exon 5.
Humans↗
Protective Effects of HLA-DRB1*08:03 and HLA-DQA1*01:03 Alleles for Alloanti-D Immunisation in the Southern Chinese D-Negative Pregnant Women.
Alloanti-D is still one of the most common causes of severe hemolytic disease of the fetus and newborn in China, as rhesus immunoglobulin (RhIG) prophylaxis is not a routine practice throughout China. HLA plays an important role in the susceptibility to alloimmunisation against red blood cell antigens. This study was designed to identify susceptible and protective HLA alleles for alloanti-D immunisation after pregnancy in the southern Chinese D-negative (D-) pregnant women. In this study, a cohort of 116 true D- pregnant females who had not received prophylactic RhIG prophylaxis, had two or more pregnancies, and did not produce alloanti-D (non-responders group), and 122 true D- pregnant women with alloanti-D immunisation (D responders group), were enrolled. HLA genotyping (HLA-A, -B, -C, -DRB1, -DPA1, -DPB1, -DQA1, and -DQB1) was performed by third generation sequencing with nanopore technology. The phenotypic frequencies of HLA alleles were compared between the D responders group and non-responders group. The results showed that the phenotypic frequencies of HLA-DRB1*08:03 and HLA-DQA1*01:03 alleles in the D responders group were significantly lower than those in the non-responders group: 1.7% versus 13.1% [Odds Ratio (OR): 0.116, 95% CI: 0.026-0.518; pc = 0.029] for HLA-DRB1*08:03 allele, and 5.2% versus 18.8% (OR: 0.235, 95% CI: 0.092-0.600, pc = 0.019) for HLA-DQA1*01:03 allele. Our findings indicated that the presence of HLA-DRB1*08:03 or HLA-DQA1*01:03 alleles can be considered as a protective factor for alloanti-D immunisation in the southern Chinese D- pregnant women.
Humans↗
Nucleic acid amplification testing and genome sequencing for WHO priority viruses in Africa: a scoping review.
Emerging viruses continue to pose serious public health threats across Africa, with recurrent outbreaks exposing gaps in diagnostics and surveillance systems. Nucleic acid amplification tests (NAATs) and genome sequencing are increasingly important for diagnostics and outbreak responses; however, their routine implementation is fragmented. This scoping review examines NAATs and genome sequencing technologies for viral detection and surveillance in Africa from 2019 to 2024, mapped to the 2024 updated WHO R&D Blueprint for Epidemics pathogen priority list. We identified 117 studies from 34 African countries reporting applications across 20 virus families, including ten designated as priorities by WHO. PCR-based assays were the most frequently reported NAATs. Illumina platforms predominated sequencing, and Oxford Nanopore Technologies were commonly used in outbreak investigations. Genome sequencing applied to priority viruses was largely reactive. NAAT-capable mobile laboratories were reported in 13 countries. Our findings underscore the need for proactive integration of NAATs into diagnostic and surveillance systems to strengthen decentralised testing, sustain genomic surveillance beyond outbreak periods, and improve early detection and preparedness for viral threats.
Journal Article↗
Assessing the readiness of Oxford Nanopore sequencing for clinical genomics applications.
Long-read sequencing (LRS) technologies, namely, Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), have emerged as promising solutions to overcome the limitations of short-read sequencing (SRS). Nevertheless, the still higher sequencing error rates compared with SRS, need for customized pipelines, rapidly updating software, and incipient scalability continue to present challenges for adopting ONT in standard clinical practice. Here we assess the performance of ONT (R9 and R10 chemistries) in comparison to Illumina and MGI across 17 well-characterized reference samples with 11 clinical variants representing nine different genetic diseases. To enable this, we have implemented a production-ready pipeline including SNV, indel, STR, SV, and CNV detection, alongside reporting key summary metrics to ensure high-quality data at the production sequencing level. Our results show high accuracy of ONT across SNVs (F-score 0.978-0.983) and SVs (F-score = 0.75) but still weaknesses across indels (F-score 0.659-0.758). However, we highlight that ONT accurately detected all four pathogenic indels as well as the performance improvement in exons and with the newer R10 chemistry. We further demonstrated the importance of long reads to detect clinically impactful variants such as a FMR1 pathogenic expansion, often misclassified by SRS as being in the premutation range. Our multiplatform analysis and Sanger validation uncovered a 1 bp error in the Coriell annotation for a cystic fibrosis-causing indel in GM07829. This work underscores the growing readiness of ONT for clinical applications, highlighting both its advancements and its potential for broader adoption in clinical genomics and large-scale operations.
Humans↗
Molecular landscape of methicillin-resistant Staphylococcus aureus strains in clinical infections from hospitals in Lagos, Nigeria.
BACKGROUND AND OBJECTIVES: Multidrug-resistant Staphylococcus aureus (MRSA) accounts for a significant proportion of antimicrobial resistance (AMR)-associated infections worldwide. This study investigated the molecular profile of MRSA in Nigeria, providing valuable genomic data to fill existing knowledge gaps and highlighting its importance in the context of the global AMR crisis. METHODS: A total of 107 isolates were obtained from patient samples, including wound swabs/pus (65 isolates, 60.7%), blood cultures (16 isolates, 15%), urine/urinary catheter (8 isolates, 7.5%) and other sources. Species identification was performed using MALDI-TOF, and antimicrobial susceptibility testing was performed using the VITEK®2 system. Genomic DNA was extracted and subjected to whole-genome sequencing using short-read Illumina technology. In addition, a subset of isolates underwent long-read sequencing using Oxford Nanopore technology. RESULTS: Among the 107 isolates, 63 (59%) were identified as MRSA, with 58 (92%) carrying the mecA gene. The MRSA isolates exhibited high resistance to non-β-lactam antibiotics, particularly trimethoprim/sulfamethoxazole (95.3%), erythromycin (76.6%), gentamicin (71.4%) and quinolones (69.8%). The most prevalent MRSA belonged to the Bengal Bay clone [t657/ST772/Staphylococcal Cassette Chromosome mec (SCCmec) V(5C2)/Panton-Valentine leukocidin (PVL) + methicillin-susceptible Staphylococcus aureus (MRSA)], followed by t4690/ST152/SCCmec Vc(5C2&5)/PVL + MRSA and ST8 (t008, n = 1; t064, n = 4)/SCCmec Vc(5C2&5). Phylogenetic analysis suggests both community/associated transmission and possible importation of strains. CONCLUSIONS: This study highlights the significant burden of MRSA in Nigeria, with the high-risk Bengal Bay MRSA clone as the most common strain. The widespread resistance to non-β-lactam antibiotics underscores the urgent need for enhanced surveillance, infection control and antibiotic stewardship to mitigate its spread.
Journal Article↗