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At least 19 recordsLinked to original sources
PubMed2025

Realfreq: real-time base modification analysis for nanopore sequencing.

SUMMARY: Nanopore sequencers allow sequencing data to be accessed in real-time. This allows live analysis to be performed, while the sequencing is running, reducing the turnaround time of the results. We introduce realfreq, a framework for obtaining real-time base modification frequencies while a nanopore sequencer is in operation. Realfreq calculates and allows access to the real-time base modification frequency results while the sequencer is running. We demonstrate that the data analysis rate with realfreq on a laptop computer can keep up with the output data rate of a nanopore MinION sequencer, while a desktop computer can keep up with a single PromethION 2 solo flowcell. AVAILABILITY AND IMPLEMENTATION: Realfreq is a free and open-source application implemented in C programming language and shell scripts. The source code and the documentation for realfreq can be found at https://github.com/imsuneth/realfreq. The version used for the manuscript is also available at https://doi.org/10.5281/zenodo.15128668.

Nanopore Sequencing↗
PubMed2026

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↗
PubMed2025

NanoFilter: enhancing phasing performance by utilizing highly consistent INDELs and SNVs in nanopore sequencing.

MOTIVATION: Nanopore sequencing data offer longer reads compared to other technologies, which is beneficial for phasing and genome assembly. INDELs provide valuable haplotype information and have significant potential to improve phasing performance. However, accurately identifying INDELs with variant callers is challenging, and incorporating INDELs into phasing remains a complex task. To address these issues, we developed NanoFilter, a novel filtering strategy designed to filter out INDELs that contain wrong phasing information based on their consistency. RESULTS: Our assessment using Nanopore R10 simplex data shows that filtering out low-consistency INDELs increases their precision from 88.3% to 98.8%, nearly matching the precision of SNVs. In the phasing results of Margin, incorporating these filtered INDELs leads to a 12.77% increase in N50 length and fewer switch errors. Furthermore, we found that SNVs filtered by NanoFilter will enhance assembly performance. When NanoFilter is integrated into the HapDup assembly pipeline, NanoFilter reduces the Hamming error rate and increases N50 length by 7.8%. AVAILABILITY AND IMPLEMENTATION: NanoFilter is available at https://github.com/Chenshanming-repo/NanoFilter (DOI: 10.5281/zenodo.16777826) and HapDup-NanoFilter is available at https://github.com/Chenshanming-repo/HapDup-NanoFilter (DOI: 10.5281/zenodo.16777890).

Nanopore Sequencing↗
PubMed2025

Leveraging basecaller's move table to generate a lightweight k-mer model for nanopore sequencing analysis.

MOTIVATION: Nanopore sequencing by Oxford Nanopore Technologies (ONT) enables direct analysis of DNA and RNA by capturing raw electrical signals. Different nanopore chemistries have varied k-mer lengths, current levels, and standard deviations, which are stored in "k-mer models." In cases where official models are lacking or unsuitable for specific sequencing conditions, tailored k-mer models are crucial to ensure precise signal-to-sequence alignment, analysis and interpretation. The process of transforming raw signal data into nucleotide sequences, known as basecalling, is a fundamental step in nanopore sequencing. RESULTS: In this study, we leverage the move table produced by ONT's basecalling software to create a lightweight de novo k-mer model for RNA004 chemistry. We demonstrate the validity of our custom k-mer model by using it to guide signal-to-sequence alignment analysis, achieving high alignment rates (97.48%) compared to larger default models. Additionally, our 5-mer model exhibits similar performance as the default 9-mer models another analysis, such as detection of m6A RNA modifications. We provide our method, termed Poregen, as a generalizable approach for creation of custom, de novo k-mer models for nanopore signal data analysis. AVAILABILITY AND IMPLEMENTATION: Poregen is an open source package under an MIT license: https://github.com/hiruna72/poregen.

Nanopore Sequencing↗
PubMed2027

Nanopore Sequencing for Chikungunya Virus: Principles and Application.

Nanopore sequencing is transforming viral genomics through real-time, portable, long-read analysis of RNA and DNA. Unlike traditional short-read platforms, it detects nucleotide sequences by measuring ionic current changes as nucleic acids pass through nanoscale pores, enabling direct single-molecule sequencing and base modification detection. Its simplicity, flexibility, and capacity for ultra-long reads make it ideal for resolving complex genomic regions, structural variants, and full viral genomes. These advantages have accelerated its use in pathogen surveillance and outbreak response, especially in resource-limited settings. For chikungunya virus (CHIKV), nanopore sequencing allows rapid, culture-independent recovery of complete genomes from clinical and vector samples, enabling real-time tracking of viral diversity, evolution, and spread. Experiences from Ebola, Zika, and COVID-19 have demonstrated the power of portable sequencing, now applied to CHIKV monitoring. Advances in tools such as Guppy, Dorado, Minimap2, and Medaka enhance read quality, consensus accuracy, and downstream analyses. Despite challenges in basecalling and error correction, robust quality control pipelines ensure reliable results. Ongoing improvements in chemistry, flow cell design, and machine learning will further enhance fidelity and throughput, establishing nanopore sequencing as a cornerstone of CHIKV genomic surveillance and epidemic preparedness.

Chikungunya virus↗
PubMed2026

Amplification-Free Nanopore Sequencing for Herpesvirus DNA Detection in Intraocular Fluids.

PURPOSE: To evaluate the feasibility of amplification-free nanopore sequencing for detecting herpesvirus DNA in intraocular fluid using multiplex polymerase chain reaction (mPCR)-characterized herpesvirus-positive and herpesvirus-negative samples. DESIGN: Retrospective, single-center, cross-sectional study. PARTICIPANTS: This study included 42 patients with uveitis whose intraocular fluid samples were examined by mPCR, including 20 mPCR-positive samples (all positive for herpesviruses) and 22 mPCR-negative samples. METHODS INTERVENTION OR TESTING: DNA extracted from intraocular fluid samples underwent ligation-based library preparation without whole-genome amplification and was sequenced on the MinION platform with Flongle flow cells for untargeted analysis. Nanopore sequencing results were compared with mPCR findings, and associations between nanopore-derived virus-specific read counts and corresponding herpesvirus DNA copy numbers measured by mPCR were assessed. MAIN OUTCOME MEASURES: Primary outcome measure was concordance between nanopore sequencing and mPCR in herpesvirus species identification. Secondary outcome measures included nanopore sequencing detection rates stratified according to mPCR-measured herpesvirus DNA copy numbers and correlations between nanopore sequencing-derived virus-specific read counts and mPCR-measured herpesvirus DNA copy numbers. RESULTS: Among 20 mPCR-positive intraocular fluid samples, nanopore sequencing identified viral DNA from the same herpesvirus species detected by mPCR in 15 (75.0%), indicating species-level concordance. None of the 22 mPCR-negative samples contained virus-specific reads. Among the 22 herpesvirus targets identified in the 20 mPCR-positive samples, herpesvirus DNA copy numbers measured by mPCR were significantly higher in nanopore-positive than in nanopore-negative targets (P = 0.015). Nanopore detection rates increased with increasing herpesvirus DNA copy numbers measured by mPCR: 3 of 6 targets (50.0%) with <105 copies/mL, 2 of 4 (50.0%) with 105-106 copies/mL, and 12 of 12 (100%) with >106 copies/mL (P = 0.021). Nanopore sequencing-derived virus-specific read counts correlated positively with herpesvirus DNA copy numbers measured by mPCR (r = 0.76, P = 0.0004). CONCLUSIONS: Amplification-free nanopore sequencing demonstrated the feasibility of detecting herpesvirus DNA in intraocular fluid samples, with detection performance dependent on herpesvirus DNA load. This simplified workflow may provide complementary information regarding viral DNA burden in minute ocular samples. FINANCIAL DISCLOSURES: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Herpesvirus↗
PubMed2025

Write and Read: Harnessing Synthetic DNA Modifications for Nanopore Sequencing.

An exciting feature of nanopore sequencing is its ability to record multi-omic information on the same sequenced DNA molecule. Well-trained models allow the detection of nucleotide-specific molecular signatures through changes in ionic current as DNA molecules translocate through the nanopore. Thus, naturally occurring DNA modifications, such as DNA methylation and hydroxymethylation, may be recorded simultaneously with the genetic sequence. Additional genomic information, such as chromatin state or the locations of bound transcription factors, may also be recorded if their locations are chemically encoded into the DNA. Here, we present a versatile "write-and-read" framework, where chemo-enzymatic DNA labeling with unnatural synthetic tags results in predictable electrical fingerprints in nanopore sequencing. As a proof-of-concept, we explore a DNA glucosylation approach that selectively modifies 5-hydroxymethylcytosine (5hmC) with glucose or glucose-azide adducts. We demonstrate that these modifications generate distinct and reproducible electrical shifts, enabling the direct detection of chemically altered nucleotides. We further demonstrate that enzymatic alkylation, such as the enzymatic transfer of azide residues to the N6 position of adenines, also produces characteristic nanopore signal shifts relative to the native adenine and 6-methyladenine. Beyond direct nucleotide detection, this approach introduces new possibilities for bio-orthogonal DNA labeling, enabling an extended alphabet of sequence-specific detectable moieties. The future use of programmable chemical modifications for simultaneous analysis of multiple omics features on individual molecules opens new avenues for genetic research and discovery.

5-hydroxymethylcytosine (5hmC)↗
PubMed2026

Evaluation of amplicon-based nanopore sequencing for foot-and-mouth disease viruses in clinical and environmental samples.

Foot-and-mouth disease (FMD) causes severe global economic loss, necessitating rapid viral characterization. Nanopore sequencing provides a simple, real-time workflow suitable for on-site outbreak response, addressing the limitations of conventional methods. In this study, we optimized a previously published amplicon-based protocol and used this method to characterize a diverse range of samples (vesicular fluid, epithelium, serum, nasal/oral swabs, and environmental samples) collected during FMD outbreaks in 2025 in the Republic of Korea. Of the 129 samples collected, we successfully recovered complete genomes from 37 samples and VP1 sequences from 85 samples. Amplifying the S-fragment in isolation and separately barcoding each pool of PCR amplicons markedly improved sequence recovery. Furthermore, sequencing success depended on viral load and sample type. Based on comparisons with real-time RT-PCR results, whole-genome sequence (WGS) recovery exceeded 77.3% at cycle threshold (Ct) values &#x2264;25 across all clinical samples. In the Ct > 30 category, serum samples yielded the highest WGS recovery rates (44.4%). This rate was markedly higher than the success rates observed for epithelium (20.0%) and nasal swabs (9.1%), whereas oral swabs and environmental samples failed to yield any sequences (0%). However, VP1 recovery from environmental samples reached 80% at Ct &#x2264; 30 (8/10), providing an approach to enable non-invasive monitoring. These findings demonstrate that amplicon-based nanopore sequencing is a practical method for the rapid generation of genomic data during FMD outbreaks.IMPORTANCEAlthough rapid detection and genomic data analysis are crucial for effective foot-and-mouth disease (FMD) control, the collection of these data can be challenging for certain sample types and impacted by reduced viral loads that result from nationwide FMD vaccination. This study provides a practical solution through large-scale evaluation of an optimized amplicon-based nanopore sequencing protocol to enhance the sequencing success rates for both clinical and environmental samples. Using a modified protocol to enhance genome recovery, we demonstrated that sequence data could be retrieved from diverse sample types (even with high real-time RT-PCR cycle threshold values). We identified serum as the most suitable sample, with environmental sample sequencing allowing for non-invasive monitoring during outbreaks. These results support the use of nanopore sequencing for rapid genomic analysis, particularly in outbreak responses, such as rapid surveillance, emergency vaccine selection, and epidemiological monitoring.

Foot-and-Mouth Disease↗
PubMed2019

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis.

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (&#x223c;26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

Cell Line↗
PubMed2025

VirDetector: a bioinformatic pipeline for virus surveillance using nanopore sequencing.

SUMMARY: Virus surveillance programmes are designed to counter the growing threat of viral outbreaks to human health. Nanopore sequencing, in particular, has proven to be suitable for this purpose, as it is readily available and provides rapid results. However, as special bioinformatic programs are required to extract the relevant information from the sequencing data, applications are needed that allow users without extensive bioinformatics knowledge to carry out the relevant analysis steps. We present VirDetector, a bioinformatic pipeline for virus surveillance using nanopore sequencing. The pipeline automatically installs all required programs and databases and allows all its steps to be executed with a single console command. After preprocessing the samples, including the possibility for basecalling, the pipeline classifies each sample taxonomically and reconstructs the viral consensus genomes, which are then used in phylogenetic analyses. This streamlined workflow provides a user-friendly and efficient solution for monitoring viral pathogens. AVAILABILITY AND IMPLEMENTATION: VirDetector is freely available at https://github.com/NLKaiser/VirDetector and https://zenodo.org/records/14637302 (10.5281/zenodo.14637302).

Nanopore Sequencing↗
PubMed2026

Rhesus blood group haplotype determination by nanopore sequencing and adaptive sampling enables the precise determination of complex allele combinations that could not be accurately determined by standard methods.

BACKGROUND: Patients with chronic transfusion needs such as those with sickle cell disease face a high risk of developing antibodies against high-prevalence antigens in the RH blood group system, complicating transfusion therapy and potentially necessitating stem cell transplantation. Molecular characterization of the RH system is hindered by hybrid alleles and high sequence homology between RHD and RHCE, limiting the effectiveness of conventional short-read sequencing. STUDY DESIGN AND METHODS: We analyzed 11 control and 20 patient samples, some of which could not be reliably genotyped by standard methods. RESULTS: Nanopore sequencing with adaptive sampling enables targeted, amplification-free long-read sequencing of the RH locus, resolving homologous and complex hybrid structures and enabling complete haplotype phasing for all samples, including samples that could not be accurately determined by standard methods like serology and short-read sequencing. Four new alleles were identified and for 13 out of 20 patients the results led to a change in the transfusion regimen. DISCUSSION: These findings show that nanopore sequencing with adaptive sampling allows unambiguous genotyping of the RH system, improves detection of complex variants, and supports better-matched transfusion strategies for chronically transfused patients.

Rh-Hr Blood-Group System↗
PubMed2025

Detection and characterization of neonatal cytomegalovirus through nanopore sequencing using flongle flow cells: Pilot study in Philadelphia, Pennsylvania.

BACKGROUND: Cytomegalovirus (CMV) remains a significant infection in neonates and its early detection can aid with further treatment (antiviral, audiology). However, current diagnostics do not provide genetic information. OBJECTIVE: We explored the use of the portable and comprehensive sequencing method from Oxford Nanopore Technologies, utilizing low-cost Flongle flow cells to detect and perform sequence-level characterization of neonatal urine samples that tested positive for CMV by PCR. STUDY DESIGN: We performed a pilot study based on a retrospective cohort study of neonates who were positive for CMV by PCR, who were admitted at two birth hospitals in Philadelphia, PA. We leveraged deep and long-read sequencing results to analyze the reads in two forms: by comparing them against a reference-based strain and by reconstructing the genome through de novo assembly with phylogenetic tree analysis. RESULTS: We assayed seven clinical samples, including a positive and negative control sample, from newborns ranging from 23 weeks' gestation to term, with testing performed for microcephaly, hearing test results, small gestational age, and thrombocytopenia. Each sample showed multiple differences compared to the reference strain, and the phylogenetic tree analysis of the de novo assembly depicted the genetic diversity of the samples. CONCLUSION: This pilot study shows that nanopore sequencing with low-cost Flongle flow cells can detect and characterize CMV strains from clinical neonatal urine samples. This, coupled with current screening and diagnostic criteria, could further our genomic understanding of neonatal CMV, such as viral genome diversity, genotype-phenotype associations, and spread of strains.

Humans↗
PubMed2026

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 &#x2264; 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 &#x2265; 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↗
PubMed2026

diffMONT: predicting methylation-specific PCR biomarkers based on nanopore sequencing data for clinical application.

MOTIVATION: DNA methylation serves as a key biomarker in clinical diagnostics, especially in cancer detection. With methylation-specific PCR (MSP), a widely used approach, patient samples can be screened fast and efficiently for differential methylation. During MSP, methylated regions are selectively amplified with specific primers. With nanopore sequencing, knowledge about DNA methylation is generated during direct DNA sequencing without needing pretreatment of the DNA. Multiple methods, mainly developed for whole-genome bisulfite sequencing (WGBS) data, exist to predict differentially methylated regions (DMRs) in the genome. However, the predicted DMRs are often very large and not sufficiently discriminating to generate meaningful results in MSP, creating a gap between theoretical cancer marker research and practical application, as no tool currently provides methylation difference predictions tailored for PCR-based diagnostics. RESULTS: Here, we present diffMONT, a tool that predicts differentially methylated regions specifically suited for MSP primer design, enabling rapid translation into practical applications. diffMONT takes into account (i) the specific length of primer and amplicon regions, (ii) the fact that one condition should be unmethylated, and (iii) a minimal required amount of differentially methylated cytosines within the primer regions. We compared the results of diffMONT to metilene and DSS based on a publicly available nanopore sequencing dataset and show that the regions predicted by diffMONT are more specific toward hypermethylated regions. diffMONT accelerates the design of methylation-specific diagnostic assays, bridging the gap between theoretical research and clinical application. AVAILABILITY AND IMPLEMENTATION: The source code for diffMONT, an open-source Python-based tool, is available at https://github.com/rnajena/diffMONT/, with an archived release under https://zenodo.org/records/17641031.

DNA Methylation↗
PubMed2024

Nanopore sequencing to detect A-to-I editing sites.

Adenosine-to-inosine (A-to-I) RNA editing, mediated by the ADAR family of enzymes, is pervasive in metazoans and functions as an important mechanism to diversify the proteome and control gene expression. Over the years, there have been multiple efforts to comprehensively map the editing landscape in different organisms and in different disease states. As inosine (I) is recognized largely as guanosine (G) by cellular machineries including the reverse transcriptase, editing sites can be detected as A-to-G changes during sequencing of complementary DNA (cDNA). However, such an approach is indirect and can be confounded by genomic single nucleotide polymorphisms (SNPs) and DNA mutations. Moreover, past studies rely primarily on the Illumina platform, which generates short sequencing reads that can be challenging to map. Recently, nanopore direct RNA sequencing has emerged as a powerful technology to address the issues. Here, we describe the use of the technology together with deep learning models that we have developed, named Dinopore (Detection of inosine with nanopore sequencing), to interrogate the A-to-I editome of any organism.

Inosine↗