TARPON-A Telomere Analysis and Research Pipeline Optimized for Nanopore.

Long-read sequencing has transformed many areas of biology and holds significant promise for telomere research by enabling analysis of nucleotide-level resolution chromosome arm–specific telomere length in both model organisms and humans. However, the adoption of new technologies, particularly in clinical or diagnostic contexts, requires careful validation to recognize potential technical and computational limitations. We present TARPON (Telomere Analysis and Research Pipeline Optimized for Nanopore), a best-practices Nextflow pipeline designed for the analysis of telomeres sequenced on the Oxford Nanopore Technologies (ONT) platform. TARPON can be executed via the command line or integrated into ONT’s EPI2ME agent, providing a user-friendly graphical interface for those without computational training. Nextflow’s container-based architecture eliminates dependency conflicts, thereby streamlining deployment across platforms. TARPON isolates telomeric repeat–containing reads, assigns strand specificity, and identifies enrichment probes that can be used both for demultiplexing and for confirming capture-based library preparation. To ensure that the analysis is restricted to full-length telomeres, reads lacking a capture probe or non-telomeric sequence on the opposite end are excluded. A sliding-window approach defines the subtelomere-to-telomere boundary, followed by quality filtering to remove low-quality or subtelomeric reads that passed earlier steps. The pipeline generates customizable statistics, text-based summaries, and publication-ready visualizations (HTML, PNG, PDF). While default settings are optimized for diagnostic workflows, all parameters are easily adjustable via the GUI or command line to support diverse applications. These include telomere analyses in variant-rich samples (e.g., ALT-positive tumors) and organisms with non-canonical telomeric repeats such as some insects (GTTAG) and certain plants (GGTTTAG). TARPON is the first complete and experimentally validated pipeline for Nanopore-based telomere analysis requiring no data pre-processing or prior bioinformatics expertise, while offering flexibility for advanced users.

Whole-genome sequencing (WGS) is an invaluable tool that enables high-resolution genotyping to precisely identify bacterial strains. It is particularly significant for highly pathogenic bacteria such as Streptococcus pneumoniae, a worldwide leading cause of mortality and morbidity. Illumina sequencing is highly established for S. pneumoniae, while Oxford Nanopore Technologies (ONT) data are limited. Hence, evaluating ONT-only data is needed. We aimed to compare the Illumina and ONT systems for S. pneumoniae sequencing. Moreover, we aimed to explore whether the newer chemistry from ONT with R10.4.1 flow cells improves the data outputs from long-read sequencing. S. pneumoniae bacteria were isolated from hospitalized patients with invasive pneumococcal disease (IPD) and serotyped by multiplex PCR. Resistance profiles were determined with anti-microbial susceptibility testing. A total of 27 isolates were sequenced using ONT Mk1c with R9.4.1 flow cells and Kit10 chemistry (ONT_V10) and the Illumina Miseq system. Illumina and ONT data were compared, and hybrid assembly was assessed. ONT sequencing was additionally performed with R10.4.1 flow cells and Kit14 chemistry (ONT_V14) in 12 isolates. S. pneumoniae identification, serotyping, AMR, and GPSC prediction were successfully achieved using ONT sequencing. The ONT_V14 chemistry significantly improved both MLST and pbp prediction in long-read sequencing. Overall, the hybrid assembly produced circular and contiguous genomes with high N50 parameters. Moreover, long-read assembly followed by short-read polishing is a fast and reliable approach for hybrid assembly at ONT sequencing depth >100×. For ONT sequencing depth <50×, tools that perform short-read-first assembly, such as Unicycler are recommended.IMPORTANCEThis study provides a detailed evaluation of whole-genome sequencing technologies and bioinformatics pipelines for the characterization of Streptococcus pneumoniae. It represents an in-depth investigation of Illumina and Oxford Nanopore technologies (ONT) systems for bacterial sequencing. It sheds light on the performance of each platform in various aspects of sequencing, including raw and assembly statistics, capsular typing, pbp typing, GPSC, AMR, and MLST prediction. This study offers a comprehensive overview of S. pneumoniae genomics and a guide for clinical and research laboratories seeking to adopt bacterial sequencing by providing important considerations when choosing sequencing platforms and analysis pipelines. We report a strong case for the implementation of WGS in the clinical setting, based on its high concordance with conventional molecular and phenotypic methods. Furthermore, the flexibility and portability of the investigated pipelines facilitate their use in clinical applications.

Introduction The human cytomegalovirus (HCMV) is a ubiquitous herpesvirus and has a complex transcriptome. Polycistronism and alternative splicing make forming accurate transcript models particularly challenging. Long-read sequencing is a powerful nover tool that is able to distinguish between isoforms and discern a complex transcriptome. In order to gain a better insight into the transcriptional repertoire of the virus, we have sequenced the lytic HCMV transcriptome on multiple third-generation sequencing platforms. Our main objectives were to determine exon-connectivity, and to annotate the lytic transcriptome of the virus. In order to utilize the power of long-read sequencing, we have developed a pipeline that is suited for the analysis of long-read RNA sequencing data and is able to compare results obtained from different sequencing platforms. We also aimed to characterize the performance of each sequencing platform and library preparation method based on their ability to sequence full-length genuine transcripts. Materials and Methods Two biologically independent samples were sequenced. The first sample was subjected to cDNA sequencing on the Pacific Biosciences (PacBio) RSII and Sequel platforms as well as cDNA and dRNA sequencing on the Oxford Nanopore Technologies (ONT) MinION platform. The second sample was used for cap-selected cDNA sequencing on the MinION platform. The data were analysed using a custom pipeline utilizing the biopython and the pysam modules, and the bedtools software. Custom scripts were written to generate read statistics, characterize transcripts and to compare results. Results Over 80,000 cDNA reads were obtained from the two PacBio platforms and over 1,000,000 cDNA reads from the MinION platform. The direct RNA sequencing yielded 36,195 reads. The direct RNA sequencing reads were used to validate the cDNA sequencing results. We have created a pipeline for the analysis of long-read RNA sequencing data which accepts mapped sequencing reads produced by any long-read sequencing platform, and outputs a transcriptome annotation based on the sequenced reads. 440 isoforms were detected in our dataset. 377 of them were novel isoforms. The novel transcripts include TSS-, TES- or alternatively spliced isoforms of known genes, antisense transcripts and a novel intergenic transcript in the short repeat region. Many of the transcript isoforms only differed from each other in a few nucleotides, however, interestingly, most isoforms differed from each other in the combination of ORFs that they contained. Discussion Our results have more than doubled the number of annotated HCMV transcripts. Cross-platform validation gives these novel features high confidence. Using long-read RNA sequencing data we were able to draw a more detailed map of the HCMV transcriptome, which is instrumental both for the analysis of the viral gene expression and for understanding the molecular mechanisms of infection. Long-read RNA sequencing has discovered countless new isoforms in all the organisms for which it has been used. The biological function of most of these isoforms is currently unknown. However, our results show that many of the isoforms have distinct coding potentials, meaning that they code for different peptides of express upstream ORFs which may play a regulatory role during translation. With the headway of long-read sequencing technologies, the importance of bioinformatics tools that can analyse such data is increasing. We developed a pipeline which can rapidly process long-read RNA sequencing data from different platforms and create a transcriptome annotation which can be utilized by user with no bioinformatics background.

Somatic SVs significantly contribute to cancer development and progression. Characterizing these variations has traditionally been difficult due to the limitations of short-read sequencing technologies and the diverse types and lengths of SVs. The advent of long-read sequencing has enabled a more comprehensive analysis of germline SVs, highlighting its potential applications in cancer genomics. The melanoma cell line COLO829, along with its normal counterpart COLO829BL, is a standard reference for somatic SV detection. Valle-Inclan et al. recently validated 68 somatic SVs combining short-read, long-read, and linked-read sequencing data. However, the sensitivity may be compromised as only a limited number of alignment-based callers were employed, such as NanoSV, Sniffles (for Oxford Nanopore Technologies (ONT)), and pbsv for (PacBio HiFi). As more alignment-based SV callers are being developed, it is crucial to integrate a broader range of tools to construct a comprehensive call set of somatic SVs. Moreover, the performance of assembly-based methods in somatic SV detection within cancer remains largely unexplored. In this study, we aimed to establish a comprehensive spectrum of somatic SVs in the COLO829 melanoma cell line by employing both alignment-based and assembly-based methods with long-read whole-genome sequencing data from PacBio and ONT aligned with Minimap2, SVs were identified and integrated from 7 alignment-based callers: cuteSV, NanoVar, DeBreak, Sniffles2, Svision-Pro, NanoSV, and pbsv. In addition, genome assemblies were constructed via Hifiasm and Verkko, incorporating HiFi reads with Hi-C and ultra-long ONT integration, followed by SV detection using four assembly-based callers: Dipcall, PAV, SVIM-asm, and cuteSV. A dedicated pipeline was developed to identify and filter somatic SVs from germline events, which considers mapping quality, sequencing depth of SV regions and their flanking regions, and 50% reciprocal overlap. Compared to the truth set established by Valle-Inclan et al., we discovered 19 novel somatic SVs, including 10 deletions, 4 insertions, 3 duplications, and 2 inversions. As a comparison, assembly-based methods detected all the deletions, insertions and duplications found by the alignment-based methods. However, it missed 20 inversions and 6 translocations. Our findings highlight the superior sensitivity of alignment-based techniques in somatic SV detection and underscore the complementary nature of assembly-based approaches. This work enhances the benchmark for somatic SV characterization in cancer genomics and confirms the utility of integrated long-read sequencing analyses. Citation Format: Zishan Peng, Zechen Chong. Full spectrum of somatic structural variations (SVs) detection in COLO829 with long-read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5056.

Complete Genome Sequences of Four Strains ofErwiniatracheiphila: A Resource for Studying aBacterial Plant Pathogen with a Highly Complex Genome.

Although many individuals with dystonia present with features indicative of single-gene etiologies, obtaining definitive genetic diagnoses can be challenging. We assessed the value of nanopore-based long-read sequencing (LRS) in achieving molecular clarification of dystonic syndromes. From a large dystonia cohort with short-read sequencing (SRS) data, 14 cases with unclear, difficult-to-evaluate, or missing causative variants were recruited. Long-read whole-genome sequencing was performed according to Oxford Nanopore Technologies (ONT) protocols. ONT sequencing produced long-range haplotypes, variant calls inaccessible to short-read technology, as well as methylation data. Phase inference allowed for changes in variant classification, establishing compound heterozygosity of causative variants in four cases. We illustrate an important advantage of LRS compared with SRS in (re)defining the identity of dystonia-causing structural variants and repeat expansions for seven individuals. One patient was found to harbor a novel exonic LINE-1 insertion in SGCE, expanding the genetic mechanism in myoclonus-dystonia. ONT data also provided unexpected insights into apparent mosaic expanded repeats in FMR1 in a subject with isolated focal dystonia. We further showed that LRS outperformed SRS in avoiding erroneous calls resulting from confounding pseudogene sequences and in discovering pathogenic alterations missed by conventional pipeline utilization (three cases). Moreover, simultaneous methylome analysis aided in directing the interpretation of three variants, including a KMT2B variant of uncertain significance that was reclassified as causal by LRS-based episignature profiling. ONT-based LRS uniquely improves analysis of dystonia-associated variations that had not previously been resolved by SRS, implying broad utility for future exploration of the molecular origins of the condition. © 2025 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The study of microbial communities has undergone significant advancements, starting from the initial use of 16S rRNA sequencing to the adoption of shotgun metagenomics. However, a new era has emerged with the advent of long-read sequencing (LRS), which offers substantial improvements over its predecessor, short-read sequencing (SRS). LRS produces reads that are several kilobases long, enabling researchers to obtain more complete and contiguous genomic information, characterize structural variations, and study epigenetic modifications. The current leaders in LRS technologies are Pacific Biotechnologies (PacBio) and Oxford Nanopore Technologies (ONT), each offering a distinct set of advantages. This review covers the workflow of long-read metagenomics sequencing, including sample preparation (sample collection, sample extraction, and library preparation), sequencing, processing (quality control, assembly, and binning), and analysis (taxonomic annotation and functional annotation). Each section provides a concise outline of the key concept of the methodology, presenting the original concept as well as how it is challenged or modified in the context of LRS. Additionally, the section introduces a range of tools that are compatible with LRS and can be utilized to execute the LRS process. This review aims to present the workflow of metagenomics, highlight the transformative impact of LRS, and provide researchers with a selection of tools suitable for this task.

Nanopore long-read RNA sequencing is reshaping extracellular vesicle (EV) research by providing the capacity to analyze full-length RNA molecules. EVs are crucial for intercellular communication, carrying a diverse range of RNA cargo that can regulate recipient cell behavior. However, traditional short-read sequencing methods involve transcript fragmentation, limiting our understanding of the EV transcriptomic landscape. Furthermore, it has been generally assumed that EV RNAs are likely to be fragmentation products of cellular RNAs, and the extent to which full length RNAs are present within EVs remains to be clarified. Recent advancements in sequencing technology, particularly long-read sequencing by Oxford Nanopore Technologies (ONT), offer a solution to this limitation. Hence, long-read sequencing allows for the analysis of full-length EV RNA molecules, providing deeper insights into their integrity and isoform diversity. Here, we present a comprehensive protocol for EV RNA purification, cDNA library preparation, and sequencing using ONT's MinION platform.

Whole-genome sequencing (WGS) for public health surveillance and epidemiological investigation of foodborne pathogens predominantly relies on sequencing platforms that generate short reads. Continuous improvement of long-read nanopore sequencing, such as Oxford nanopore technologies (ONT), presents a potential for leveraging multiple advantages of the technology in public health and food industry settings, including rapid turnaround and onsite applicability in addition to superior read length. Using an established cohort of Salmonella Enteritidis isolates for subtyping evaluation, we assessed the technical readiness of nanopore long read sequencing for single nucleotide polymorphism (SNP) analysis and core-genome multilocus sequence typing (cgMLST) of a major foodborne pathogen. By multiplexing three isolates per flow cell, we generated sufficient sequencing depths in <7 h of sequencing for robust subtyping. SNP calls by ONT and Illumina reads were highly concordant despite homopolymer errors in ONT reads (R9.4.1 chemistry). In silico correction of such errors allowed accurate allelic calling for cgMLST and allelic difference measurements to facilitate heuristic detection of outbreak isolates. IMPORTANCE Evaluation, standardization, and implementation of the ONT approach to WGS-based, strain-level subtyping is challenging, in part due to its relatively high base-calling error rates and frequent iterations of sequencing chemistry and bioinformatic analytics. Our study established a baseline for the continuously evolving nanopore technology as a viable solution to high-quality subtyping of Salmonella, delivering comparable subtyping performance when used standalone or together with short-read platforms. This study paves the way for evaluating and optimizing the logistics of implementing the ONT approach for foodborne pathogen surveillance in specific settings.

Long-read sequencing -Pitfalls, benefits and success stories Long-read sequencing is an approach that holds promise to obtain genomic information for the first time in its entirety, accurately and resolved by haplotypes. In recent years the accuracy of long-read sequencing techniques, such as Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), has significantly improved. Already, long-read sequencing can be used to reach an unprecedented genetic resolution (Garg, 2021), e.g. by covering previously inaccessible genomic regions or entire RNA transcripts in individual reads. Hence, long-read sequencing is a game changer in genetic research for many fields. Advancements in sequencing technology require major, simultaneous algorithmic developments, for example, in the context of base calling, variant calling and assembly. The latest ONT Q20+ and PacBio high fidelity (HiFi) protocols have revolutionized sequencing by producing reads with lengths in the order of tens of kilobases up to megabases and with base accuracies exceeding 99% (Logsdon et al., 2020) . While the HiFi technology produces relatively more accurate sequences than ONT, ONT can generate ultra-long reads and is scalable from portable devices to benchtop sequencers. The key requirement of both technologies is high molecular weight DNA. Rapidly decreasing sequencing costs have fueled the generation of increasing amounts of genomic data for biodiversity applications and human health (De Coster et al., 2021; Miller et al., 2021) . An example is the implementation of next-generation sequencing (NGS) into clinical practice for diagnosis, prognosis, and therapy selection in various medical fields, of which oncology has been a pioneer (Berger and Mardis, 2018) . Undoubtedly, challenges still exist, such as input material requirements and establishing robust data analysis methods. Further, extensive method development and benchmarking are needed and ongoing to fully unlock the potential of long reads for these and other applications (Amarasinghe et al., 2020) .

Background Genome structural variants (SVs) have larger effect on human genome functions than single nucleotide variants (SNVs). Although short-read sequencing (SRS) is current major next generation sequencing method and has given us a great benefit to elucidate the genetic background of inherited diseases, it does not detect SVs accurately. Long-read sequencing (LRS) produces tens to thousands of kilobases reads and detects the breakpoints of complex SVs. This study aimed to confirm a large deletion, which was suspected by SRS, using LRS by Oxford Nanopore technology (ONT). Methods Genomic libraries for SRS was prepared with HaloPlex. Targeted SRS was performed for 58 genes with MiSeq. Genomic libraries for LRS were prepared using the Ligation sequencing 1D kit SQK-LSK109 (ONT). Whole genome LRS was performed with GridION X5 and R9.4 flow cells (ONT). Results The patient was a five-month-old boy with atrial septal defect (ASD) and atrial tachycardia. Though SRS failed to identify any causative SNVs, the results with SureCall software (Agilent) suspected a deletion between exon 3 to exon 26 in MYH6 encoding α heavy chains of cardiac myosin. The variants in MYH6 are known to be associated with ASD. Because a deletion between MYH6 exon 26 and MYH7 exon 27 was reported as esv2748480 on the Database of Genomic Variants, we performed long-range PCR from MYH6 intron26 to MYH7 exon26 and found an abnormal 1.5K bases PCR product only in the case. Due to high homology of MYH6 and MYH7, Sanger sequencing failed to detect the break point. In LRS, 3 flow cells generated 3.8M base-called reads containing 42G bases with N50 of 13K bases. We used NGMLR, which is a long-read mapper, to align the reads to the human reference genome (hg38). SVs were called by Sniffles detecting all types of SVs. The deletion was found to range from chr14: 23390037 to 23419824 (see figure) and did not contain other SVs. There was no pathogenic SV on ACTC1, GATA4, TBX20 and TLL1 which are genes related to ASD on Genetic Testing Registry. His mother had also ASD and harbored the same deletion. Conclusions This is the first report to identify a large deletion between MYH6 and MYH7 in the family with ASD. The combination of SRS and LRS is useful to detect SVs in patients with suspected inherited diseases but carried no causative SNVs. Funding Acknowledgement Type of funding source: None

Combining panel-based and whole-transcriptome-based gene fusion detection by long-read sequencing.

Ten years ago, the journal Molecular Ecology published a “road map” paper that reviewed past achievements in the discipline of molecular ecology, identified research challenges and charted a way forward (Andrew et al., 2013). That paper was motivated by a symposium organized during the First Joint Congress on Evolutionary Biology (Ottawa, July 6–10, 2012). In addition, it occurred on the heels of a major inflection point in molecular ecology and in life sciences more broadly: the development and uptake of “next”- or “second”-generation sequencing technologies, which deliver short DNA reads (typically shorter than 400 bp) at very high throughput (e.g., several billion reads per run; Goodwin et al., 2016). As such, Andrew et al. (2013) emphasized the promise of second-generation sequencing for diverse subdisciplines of molecular ecology such as phylogeography, landscape genomics, molecular adaptation and speciation. Representing more than just a technical advancement, second-generation sequencing was predicted to stimulate rapid conceptual breakthroughs in the field, especially in nonmodel species (Stapley et al., 2010; Tautz et al., 2010). As illustrated by any recent issue in the Molecular Ecology journal, these predictions were accurate.

High-throughput massive parallel sequencing has significantly improved bacterial pathogen genomics, diagnostics, and epidemiology. Despite its high accuracy, short-read sequencing struggles with the complete genome reconstruction and assembly of extrachromosomal elements such as plasmids. Long-read sequencing with Oxford Nanopore Technologies (ONT) presents an alternative that offers benefits including real-time sequencing and cost efficiency, particularly useful in resource-limited settings. However, the historically higher error rates of ONT data have so far limited its application in high-precision genomic typing. The recent release of ONT's R10.4.1 chemistry, with significantly improved raw read accuracy (Q20+), offers a potential solution to this problem. The aim of this study is to evaluate the performance of ONT's latest chemistry for bacterial genomic typing against the gold-standard Illumina technology, focusing on three respiratory pathogens of public health importance, Klebsiella pneumoniae, Bordetella pertussis, and Corynebacterium diphtheriae, and their related species. Using the Rapid Barcoding Kit V14, we generate and analyze genome assemblies with different basecalling models, at different simulated depths of coverage. ONT assemblies are compared to the Illumina reference for completeness and core genome multilocus sequence typing (cgMLST) accuracy (number of allelic mismatches). Our results show that genomes obtained from raw ONT data basecalled with Dorado SUP v0.9.0, assembled with Flye, and with a minimum coverage depth of 35×, optimized accuracy for all bacterial species tested. Error rates are consistently <0.5% for each cgMLST scheme, indicating that ONT R10.4.1 data are suitable for high-resolution genomic typing applied to outbreak investigations and public health surveillance.

Description Sequencing antigen-specific T cell receptors (TCR) remains a major technologic challenge, requiring labor- and cost-intensive single-cell approaches. We have developed a rapid and economical method for sequencing TCR mRNA from antigen stimulated peripheral blood mononuclear cells using targeted long-read nanopore sequencing with Oxford Nanopore Technologies’ (ONT) Minion sequencer. Targeted PCR amplification of TCR alpha and beta cDNA was performed with a template switching oligo (TSO). Dual unique molecular identifiers (UMI) were used for single cell, oligoclonal, or bulk cell sample specific sequence identification followed by nanopore sequencing. The reads were demultiplexed by UMIs and aligned to germline V,D, and J gene sequences from the IMGT database for identification. Error corrected sequences were then used to determine the non-templated complementarity determining region 3 (CDR3) sequence for the TCRs. From bulk PBMCs, TCR Beta Chain had 796 unique TRBV-TRBJ combination and TCR Alpha Chain had 2053 unique TRAV-TRAJ combinations. Human PBMC samples were spiked with 10% Jurkat cells and sequenced using this method for 12 hours resulting in 200K reads. After read demultiplexing, filtering (Q &amp;gt; 25), and alignment, it was found that 8.8% of TCR beta and 7.0% TCR alpha reads matched the Jurkat TCR sequence in the 10% spiked sample. This method provides a fast and cost-effective approach to generating antigen specific TCR sequences for downstream functional studies. Funding Sources Supported by NIH/NCI 5U01CA281660 Topic Categories Technological Innovations in Immunology (TECH)