Long-read sequencing data analysis for yeasts.

BackgroundThe barred knifejaw (Oplegnathus fasciatus), a member of the Oplegnathidae family of the Centrarchiformes, is a commercially important rocky reef fish native to East Asia. Oplegnathus fasciatus has become an important fishery resource for offshore cage aquaculture and fish stocking of marine ranching in China, Japan, and Korea. Recently, sexual dimorphism in growth with neo-sex chromosome and widespread biotic diseases in O. fasciatus have been increasing concern in the industry. However, adequate genome resources for gaining insight into sex-determining mechanisms and establishing genetically resistant breeding systems for O. fasciatus are lacking. Here, we analyzed the entire genome of a female O. fasciatus fish using long-read sequencing and Hi-C data to generate chromosome-length scaffolds and a highly contiguous genome assembly.FindingsWe assembled the O. fasciatus genome with a total of 245.0 Gb of raw reads that were generated using both Pacific Bioscience (PacBio) Sequel and Illumina HiSeq 2000 platforms. The final draft genome assembly was approximately 778.7 Mb, which reached a high level of continuity with a contig N50 of 2.1 Mb. The genome size was consistent with the estimated genome size (777.5 Mb) based on k-mer analysis. We combined Hi-C data with a draft genome assembly to generate chromosome-length scaffolds. Twenty-four scaffolds corresponding to the 24 chromosomes were assembled to a final size of 768.8 Mb with a contig N50 of 2.1 Mb and a scaffold N50 of 33.5 Mb using 1,372 contigs. The identified repeat sequences accounted for 33.9% of the entire genome, and 24 003 protein-coding genes with an average of 10.1 exons per gene were annotated using de novo methods, with RNA sequencing data and homologies to other teleosts. According to phylogenetic analysis using protein-coding genes, O. fasciatus is closely related to Larimichthys crocea, with O. fasciatus diverging from their common ancestor approximately 70.5–88.5 million years ago.ConclusionsWe generated a high-quality draft genome for O. fasciatus using long-read PacBio sequencing technology, which represents the first chromosome-level reference genome for Oplegnathidae species. Assembly of this genome assists research into fish sex-determining mechanisms and can serve as a resource for accelerating genome-assisted improvements in resistant breeding systems.

Cellular genetic heterogeneity is common in many biological conditions including cancer, microbiome, and co-infection of multiple pathogens. Detecting and phasing minor variants play an instrumental role in deciphering cellular genetic heterogeneity, but they are still difficult tasks because of technological limitations. Recently, long-read sequencing technologies, including those by Pacific Biosciences and Oxford Nanopore, provide an opportunity to tackle these challenges. However, high error rates make it difficult to take full advantage of these technologies. To fill this gap, we introduce iGDA, an open-source tool that can accurately detect and phase minor single-nucleotide variants (SNVs), whose frequencies are as low as 0.2%, from raw long-read sequencing data. We also demonstrate that iGDA can accurately reconstruct haplotypes in closely related strains of the same species (divergence ≥0.011%) from long-read metagenomic data.

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.

Long-read sequencing (LRS) technologies have the potential to revolutionize scientific discoveries in RNA biology through the comprehensive identification and quantification of full-length mRNA isoforms. Despite great promise, challenges remain in the widespread implementation of LRS technologies for RNA-based applications, including concerns about low coverage, high sequencing error, and robust computational pipelines. Although much focus has been placed on defining mRNA exon composition and structure with LRS data, less careful characterization has been done of the ability to assess the terminal ends of isoforms, specifically, transcription start and end sites. Such characterization is crucial for completely delineating full mRNA molecules and regulatory consequences. However, there are substantial inconsistencies in both start and end coordinates of LRS reads spanning a gene, such that LRS reads often fail to accurately recapitulate annotated or empirically derived terminal ends of mRNA molecules. Here, we describe the specific challenges of identifying and quantifying mRNA terminal ends with LRS technologies and how these issues influence biological interpretations of LRS data. We then review recent experimental and computational advances designed to alleviate these problems, with ideal use cases for each approach. Finally, we outline anticipated developments and necessary improvements for the characterization of terminal ends from LRS data.

BackgroundLong-read sequencing can be applied to generate very long contigs and even completely assembled genomes at relatively low cost and with minimal sample preparation. As a result, long-read sequencing platforms are becoming more popular. In this respect, the Oxford Nanopore Technologies–based long-read sequencing “nanopore" platform is becoming a widely used tool with a broad range of applications and end-users. However, the need to explore and manipulate the complex data generated by long-read sequencing platforms necessitates accompanying specialized bioinformatics platforms and tools to process the long-read data correctly. Importantly, such tools should additionally help democratize bioinformatics analysis by enabling easy access and ease-of-use solutions for researchers.ResultsThe Galaxy platform provides a user-friendly interface to computational command line–based tools, handles the software dependencies, and provides refined workflows. The users do not have to possess programming experience or extended computer skills. The interface enables researchers to perform powerful bioinformatics analysis, including the assembly and analysis of short- or long-read sequence data. The newly developed “NanoGalaxy" is a Galaxy-based toolkit for analysing long-read sequencing data, which is suitable for diverse applications, including de novo genome assembly from genomic, metagenomic, and plasmid sequence reads.ConclusionsA range of best-practice tools and workflows for long-read sequence genome assembly has been integrated into a NanoGalaxy platform to facilitate easy access and use of bioinformatics tools for researchers. NanoGalaxy is freely available at the European Galaxy server https://nanopore.usegalaxy.eu with supporting self-learning training material available at https://training.galaxyproject.org.

With the increasing affordability and accessibility of genome sequencing data, de novo genome assembly is an important first step to a wide variety of downstream studies and analyses. Therefore, bioinformatics tools that enable the generation of high-quality genome assemblies in a computationally efficient manner are essential. Recent developments in long-read sequencing technologies have greatly benefited genome assembly work, including scaffolding, by providing long-range evidence that can aid in resolving the challenging repetitive regions of complex genomes. ntLink is a flexible and resource-efficient genome scaffolding tool that utilizes long-read sequencing data to improve upon draft genome assemblies built from any sequencing technologies, including the same long reads. Instead of using read alignments to identify candidate joins, ntLink utilizes minimizer-based mappings to infer how input sequences should be ordered and oriented into scaffolds. Recent improvements to ntLink have added important features such as overlap detection, gap-filling, and in-code scaffolding iterations. Here, we present three basic protocols demonstrating how to use each of these new features to yield highly contiguous genome assemblies, while still maintaining ntLink's proven computational efficiency. Further, as we illustrate in the alternate protocols, the lightweight minimizer-based mappings that enable ntLink scaffolding can also be utilized for other downstream applications, such as misassembly detection. With its modularity and multiple modes of execution, ntLink has broad benefit to the genomics community, from genome scaffolding and beyond. ntLink is an open-source project and is freely available from https://github.com/bcgsc/ntLink. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: ntLink scaffolding using overlap detection Basic Protocol 2: ntLink scaffolding with gap-filling Basic Protocol 3: Running in-code iterations of ntLink scaffolding Alternate Protocol 1: Generating long-read to contig mappings with ntLink Alternate Protocol 2: Using ntLink mappings for genome assembly correction with Tigmint-long Support Protocol: Installing ntLink.

As an economically important plant parasitic nematode (PPN), Heterodera filipjevi causes great damage on wheat, and now it was widely recorded in many countries. While multiple genomes of PPNs have been published, high-quality genome assembly and annotation on H. filipjevi have yet to be performed. This study presents a chromosome-scale genome assembly and annotation for H. filipjevi, utilizing a combination of Illumina short-read, PacBio long-read, and Hi-C sequencing technologies. The genome consists of 9 pseudo-chromosomes that contain 134.19 Mb of sequence, with a scaffold N50 length of 11.88 Mb. In total, 10,036 genes were annotated, representing 75.20% of the total predicted protein-coding genes. Our study provides the first chromosome-scale genome for H. filipjevi, which is also the inaugural high-quality genome of cereal cyst nematodes (CCNs). It provides a valuable genomic resource for further biological research and pest management of cereal cyst nematodes disease.

BackgroundGenomic data have become major resources to understand complex mechanisms at fine-scale temporal and spatial resolution in functional and evolutionary genetic studies, including human diseases, such as cancers. Recently, a large number of whole genomes of evolving populations of yeast (Saccharomyces cerevisiae W303 strain) were sequenced in a time-dependent manner to identify temporal evolutionary patterns. For this type of study, a chromosome-level sequence assembly of the strain or population at time zero is required to compare with the genomes derived later. However, there is no fully automated computational approach in experimental evolution studies to establish the chromosome-level genome assembly using unique features of sequencing data.Methods and resultsIn this study, we developed a new software pipeline, the integrative meta-assembly pipeline (IMAP), to build chromosome-level genome sequence assemblies by generating and combining multiple initial assemblies using three de novo assemblers from short-read sequencing data. We significantly improved the continuity and accuracy of the genome assembly using a large collection of sequencing data and hybrid assembly approaches. We validated our pipeline by generating chromosome-level assemblies of yeast strains W303 and SK1, and compared our results with assemblies built using long-read sequencing and various assembly evaluation metrics. We also constructed chromosome-level sequence assemblies of S. cerevisiae strain Sigma1278b, and three commonly used fungal strains: Aspergillus nidulans A713, Neurospora crassa 73, and Thielavia terrestris CBS 492.74, for which long-read sequencing data are not yet available. Finally, we examined the effect of IMAP parameters, such as reference and resolution, on the quality of the final assembly of the yeast strains W303 and SK1.ConclusionsWe developed a cost-effective pipeline to generate chromosome-level sequence assemblies using only short-read sequencing data. Our pipeline combines the strengths of reference-guided and meta-assembly approaches. Our pipeline is available online at http://github.com/jkimlab/IMAP including a Docker image, as well as a Perl script, to help users install the IMAP package, including several prerequisite programs. Users can use IMAP to easily build the chromosome-level assembly for the genome of their interest.

Neurodegenerative diseases exhibit chronic progressive lesions in the central and peripheral nervous systems with unclear causes. The search for pathogenic mutations in human neurodegenerative diseases has benefited from massively parallel short-read sequencers. However, genomic regions, including repetitive elements, especially with high/low GC content, are far beyond the capability of conventional approaches. Recently, long-read single-molecule DNA sequencing technologies have emerged and enabled researchers to study genomes, transcriptomes, and metagenomes at unprecedented resolutions. The identification of novel mutations in unresolved neurodegenerative disorders, the characterization of causative repeat expansions, and the direct detection of epigenetic modifications on naive DNA by virtue of long-read sequencers will further expand our understanding of neurodegenerative diseases. In this article, we review and compare 2 prevailing long-read sequencing technologies, Pacific Biosciences and Oxford Nanopore Technologies, and discuss their applications in neurodegenerative diseases.

Motivationde novo variants (DNVs) are variants that are present in offspring but not in their parents. DNVs are both important for examining mutation rates as well as in the identification of disease-related variation. While efforts have been made to call DNVs, calling of DNVs is still challenging from parent–child sequenced trio data. We developed Hare And Tortoise (HAT) as an automated DNV detection workflow for highly accurate short-read and long-read sequencing data. Reliable detection of DNVs is important for human genomics and HAT addresses this need.ResultsHAT is a computational workflow that begins with aligned read data (i.e. CRAM or BAM) from a parent–child sequenced trio and outputs DNVs. HAT detects high-quality DNVs from Illumina short-read whole-exome sequencing, Illumina short-read whole-genome sequencing, and highly accurate PacBio HiFi long-read whole-genome sequencing data. The quality of these DNVs is high based on a series of quality metrics including number of DNVs per individual, percent of DNVs at CpG sites, and percent of DNVs phased to the paternal chromosome of origin.Availability and implementationhttps://github.com/TNTurnerLab/HAT

BackgroundThe recognition that transposable elements (TEs) play important roles in many biological processes has elicited growing interest in analyzing sequencing data derived from this ‘dark genome’. This goal is complicated by the highly repetitive nature of these sequences in genomes, requiring the deployment of several problem-specific tools as well as the curation of appropriate genome annotations. This pipeline aims to make the analysis of TE sequences and their expression more generally accessible.ResultsThe TE-Seq pipeline conducts an end-to-end analysis of RNA sequencing data, examining both genes and TEs, and is compatible with most eukaryotic species. It implements computational methods tailor-made for TEs, and produces a comprehensive analysis of TE expression at both the level of the individual element and at the TE clade level. Furthermore, if supplied with long-read DNA sequencing data, it is able to assess TE expression from non-reference (polymorphic) loci. As a demonstration, we analyzed proliferating, early senescent, and late senescent human lung fibroblast RNA-Seq data, and created a custom reference genome and annotations for this cell strain using Nanopore sequencing data. We found that several retrotransposable element clades were upregulated in senescence, which included non-reference, intact, and potentially active elements.ConclusionsTE-Seq is made available as a Snakemake pipeline which can be obtained at https://github.com/maxfieldk/TE-Seq.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00381-w.

Modbed track: Visualization of modified bases in single-molecule sequencing

Long-read sequencing (LRS) technologies have enhanced the ability to resolve complex genomic architecture and determine the ‘phase’ relationships of genetic variants over long distances. Although genome-wide association studies (GWAS) identify individual variants associated with complex traits, they do not typically account for whether multiple associated signals at a locus may act in cis or trans, or whether they reflect allelic heterogeneity. As a result, effects that arise specifically from phase relationships may remain hidden in analyses using short-read and microarray data. While the advent of LRS has enabled accurate measurement of phase in population cohorts, statistical methods that leverage phase in genetic association analysis remain underdeveloped. Here, we introduce the Regression on Phase (RoP) method, which directly models cis and trans phase effects between variants under a regression framework. In simulations, RoP outperforms genotype interaction tests that detect phase effects indirectly, and distinguishes in-cis from in-trans phase effects. We implemented RoP at two cystic fibrosis (CF) modifier loci discovered by GWAS. At the chromosome 7q35 trypsinogen locus, RoP confirmed that two variants contributed independently (allelic heterogeneity). At the SLC6A14 locus on chromosome X, phase analysis uncovered a coordinated regulatory mechanism in which a promoter variant modulates lung phenotypes in individuals with CF when acting in cis with a lung-specific enhancer (E2765449/enhD). This coordinated regulation was confirmed in functional studies. These findings highlight the potential of leveraging phase information from LRS in genetic association studies. Analyzing phase effects with RoP can provide deeper insights into the complex genetic architectures underlying disease phenotypes, ultimately guiding more informed functional investigations and potentially revealing new therapeutic targets.

Tea gray blight is one of the most serious foliar diseases of tea tree, caused by the plant-pathogenic fungus Pseudopestalotiopsis theae, which can affect production and quality of tea worldwide. We generated a highly contiguous, 50.41-Mbp genome assembly (N50 = 1.30 Mbp) of P. theae strain CYF27 by combining PacBio long-read and Illumina short-read sequencing technologies. We identified a total of 15,626 gene models, of which 1,038 genes encode putative secreted proteins. The high-quality genome assembly and annotation resource reported here will be useful for the study of fungal infection mechanisms and pathogen-host interaction.

The cottids (Cottidae) are a taxonomically diverse and ecologically important component of many marine and freshwater ecosystems. Despite recent breakthroughs in long-read sequencing, high quality genomic resources are still limited for studies of ecological and evolutionary processes in cottids. Here, we generated a high-quality, chromosome-scale genome assembly (521.26Mb) of the catadromous roughskin sculpin (Trachidermus fasciatus Heckel) with a contig N50 of 2.93Mb and a scaffold N50 of 24.06Mb. Approximately 21.97% of the genome was composed of repetitive elements. A total of 21,872 protein-coding genes were predicted, of which 19,900genes (90.98%) were functionally annotated. Phylogenetic analysis supported the validity of Scorpaenoidei and Cottioidei as two suborders of the Perciformes. Chromosome-scale collinearity analyses identified four chromosome fusions leading to the reduction of chromosome number in T. fasciatus. Gene families related to cell apoptosis and cell death were expanded and those related to immune system were contracted, suggesting that these gene families might be relevant to a host of phenotypic differences between T. fasciatus and other teleosts. Gene families associated with osmoregulation were also expanded, which might be associated with its catadromous life history. A total of 50 aging-associated genes were found to be under positive selection, which might be associated with the short lifespan of T. fasciatus. The high-quality genome assembly and annotation will promote researches into the evolution of catadromous life history and short lifespan for T. fasciatus and facilitate comparative genomic studies of cottids in the near future.