Long-read Sequencing Research Articles

Widespread 3′UTR capped RNAs derive from G-rich regions in proximity to AGO2 binding sites

Abstract The 3′ untranslated region (3′UTR) plays a crucial role in determining mRNA stability, localisation, translation and degradation. Cap analysis of gene expression (CAGE), a method for the detection of capped 5′ ends of mRNAs, additionally reveals a large number of apparently 5′ capped RNAs derived from locations within the body of the transcript, including 3′UTRs. Here, we provide direct evidence that these 3′UTR-derived RNAs are indeed capped and widespread in mammalian cells. By using a combination of AGO2 enhanced individual nucleotide resolution UV crosslinking and immunoprecipitation (eiCLIP) and CAGE following siRNA treatment, we find that these 3′UTR-derived RNAs likely originate from AGO2-binding sites, and most often occur at locations with G-rich motifs bound by the RNA-binding protein UPF1. High-resolution imaging and long-read sequencing analysis validate several 3′UTR-derived RNAs, showcase their variable abundance and show that they may not co-localise with the parental mRNAs. Taken together, we provide new insights into the origin and prevalence of 3′UTR-derived RNAs, show the utility of CAGE-seq for their genome-wide detection and provide a rich dataset for exploring new biology of a poorly understood new class of RNAs. Graphical Abstract Schematic representation of the proposed model where 3′UTR-derived RNAs originate from G-rich regions enriched in AGO2 and UPF1 binding sites.

Chromosome-level reference genome for the Jonah crab, Cancer borealis.

The Jonah crab, Cancer borealis, is integral to marine ecosystems and supports a rapidly growing commercial fishery in the northwest Atlantic Ocean. This species also has a long history as a model for neuroscience that has expanded our understanding of central pattern generators, neuromodulation, synaptic plasticity, and the connectivity of neural circuits. Here we present a highly contiguous reference genome for the Jonah crab that will provide an essential resource to advance fisheries, conservation, and biomedical research. Using a combination of PacBio long-read sequencing and Omni-C scaffolding, we generated a final genome assembly spanning 691 Mb covering 51 chromosome-length scaffolds and 106 additional contigs. Benchmarking Universal Single-Copy Ortholog (BUSCO) analysis indicated a high-quality assembly with a completeness score of 90.8%. Repeat annotation identified 1,649 repeat families making up 48.27% of the Jonah crab genome. Gene model predictions annotated 24,830 protein coding genes with a 92.3% BUSCO score. Gene family evolution analysis revealed the expansion of gene families associated with nervous system function, and targeted analysis revealed an extensive repertoire of neural genes. The Jonah crab genome will not only provide a resource for neuroscience research but will also serve as a foundation to investigate adaptation to stress and population structure to support sustainable fisheries management during this time of rapidly changing environmental conditions in the northwest Atlantic Ocean.

A genome assembly for the California endemic liverwort Calasterella californica.

Calasterella californica belongs to a monotypic genus of liverworts endemic to the west coast of North America, primarily distributed in California. This dioicous species occurs in a variety of ecosystems from deserts to redwood forest; little is known about how this species is adapted to live in those seemingly contrasting environments. In this paper, we report the assembly of the nuclear genome of Calasterella californica. As part of the California Conservation Genomics Project (CCGP), we used Pacific Biosciences HiFi long-read sequencing data to produce a de novo assembly that consists of 772 contigs, with a total length of 517 Mbp and a BUSCO complete score of 95%. C. californica is only the sixth species of liverworts - a group with more than 7200 described species - to have a nuclear reference genome. The availability of this reference genome will facilitate the study of the unique features of C. californica and other liverworts, pave the road towards a comparative understanding of liverwort genomes, and add an important starting point for studies of the geographic variation of this species within the CCGP project.

Factors impacting target-enriched long-read sequencing of resistomes and mobilomes.

We investigated the efficiency of target-enriched long-read sequencing (TELSeq) for detecting antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) within complex matrices. We aimed to overcome limitations associated with traditional antimicrobial resistance (AMR) detection methods, including short-read shotgun metagenomics, which can lack sensitivity, specificity, and the ability to provide detailed genomic context. By combining biotinylated probe-based enrichment with long-read sequencing, we facilitated the amplification and sequencing of ARGs, eliminating the need for bioinformatic reconstruction. Our experimental design included replicates of human fecal microbiota transplant material, bovine feces, pristine prairie soil, and a mock human gut microbial community, allowing us to examine variables including genomic DNA input and probe set composition. Our findings demonstrated that TELSeq markedly improves the detection rates of ARGs and MGEs compared to traditional sequencing methods, underlining its potential for accurate AMR monitoring. A key insight from our research is the importance of incorporating mobilome profiles to better predict the transferability of ARGs within microbial communities, prompting a recommendation for the use of combined ARG-MGE probe sets for future studies. We also reveal limitations for ARG detection from low-input workflows, and describe the next steps for ongoing protocol refinement to minimize technical variability and expand utility in clinical and public health settings. This effort is part of our broader commitment to advancing methodologies that address the global challenge of AMR.

Best practices for germline variant and DNA methylation analysis of second- and third-generation sequencing data.

This comprehensive review provides insights and suggested strategies for the analysis of germline variants using second- and third-generation sequencing technologies (SGS and TGS). It addresses the critical stages of data processing, starting from alignment and preprocessing to quality control, variant calling, and the removal of artifacts. The document emphasized the importance of meticulous data handling, highlighting advanced methodologies for annotating variants and identifying structural variations and methylated DNA sites. Special attention is given to the inspection of problematic variants, a step that is crucial for ensuring the accuracy of the analysis, particularly in clinical settings where genetic diagnostics can inform patient care. Additionally, the document covers the use of various bioinformatics tools and software that enhance the precision and reliability of these analyses. It outlines best practices for the annotation of variants, including considerations for problematic genetic alterations such as those in the human leukocyte antigen region, runs of homozygosity, and mitochondrial DNA alterations. The document also explores the complexities associated with identifying structural variants and copy number variations, underscoring the challenges posed by these large-scale genomic alterations. The objective is to offer a comprehensive framework for researchers and clinicians, ensuring that genetic analyses conducted with SGS and TGS are both accurate and reproducible. By following these best practices, the document aims to increase the diagnostic accuracy for hereditary diseases, facilitating early diagnosis, prevention, and personalized treatment strategies. This review serves as a valuable resource for both novices and experts in the field, providing insights into the latest advancements and methodologies in genetic analysis. It also aims to encourage the adoption of these practices in diverse research and clinical contexts, promoting consistency and reliability across studies.

The quality and detection limits of mitochondrial heteroplasmy by long read nanopore sequencing

This study evaluates long-read and short-read sequencing for mitochondrial DNA (mtDNA) heteroplasmy detection. 592,315 bootstrapped datasets generated from two single-nucleotide mismatched ultra-deep sequenced mtDNA samples were used to assess basecalling error and accuracy, limit of heteroplasmy detection, and heteroplasmy detection across various coverage depths. Results showed high Phred scores of data with GC-rich sequence bias for long reads. Limit of detection of 12% heteroplasmy was identified, showing strong correlation (R2 ≥ 0.955) with expected heteroplasmy but underreporting tendency of high-level variants. Nanopore sequencing shows potential for direct applicability in mitochondrial diseases diagnostics, but stringent validation processes to ensure diagnostic result quality are required.

ITSxpress version 2: software to rapidly trim internal transcribed spacer sequences with quality scores for amplicon sequencing.

The nuclear ribosomal RNA (rRNA) internal transcribed spacer (ITS) regions are commonly used to identify fungi and other eukaryotic taxa in amplicon sequencing. The highly conserved rRNA regions flanking the ITS are often trimmed before being used for taxonomic assignment. The Python software package ITSxpress rapidly trims single-end or paired-end sequences in FASTQ format for use in amplicon sequence variant clustering methods like DADA2. This new major release of ITSxpress improves the paired-end merging method, simplifies installation of the QIIME 2 ITSxpress plugin, removes major dependencies, adds use cases, and is compatible with newer compression formats. This article discusses the modifications to ITSxpress that improve the output and user experience, leading to a major version increase.IMPORTANCEITSxpress is a sequence trimming method applied to internal transcribed spacer (ITS) amplicon sequences before calling amplicon sequence variants (ASVs). The ITS region is used to understand the composition of eukaryotic microbial communities. ITS sequences provide good taxonomic resolution due to their hypervariability, but are flanked by conserved regions that allow their primers to be more universal. Amplicons generated with such primers contain regions with different evolutionary rates, and trimming these conserved regions results in better taxonomic classification and a more valid set of ASVs. This package can be used for most amplicon sequencing methods including for newer long-read sequencing formats, such as PacBio. ITSxpress can be installed from Bioconda, used as a Docker image, or installed from source code. The package works well with high-performance computing clusters or laptops due to its low-resource requirements.

A chromosome-level genome assembly of a model conifer plant, the Japanese cedar, Cryptomeria japonica D. Don.

The Japanese cedar (Cryptomeria japonica D. Don) is one of the most important Japanese forest trees, occupying approximately 44% of artificial forests and planted in East Asia, the Azores Archipelago, and certain islands in the Indian Ocean. Although the huge genome of the species (ca. 9 Gbp) with abundant repeat elements may have represented an obstacle for genetic analysis, this species is easily propagated by cutting, flowered by gibberellic acid, transformed by Agrobacterium, and edited by CRISPR/Cas9. These characteristics of C. japonica recommend it as a model conifer species for which reference genome sequences are necessary. Herein, we report the first chromosome-level assembly of C. japonica (2n = 22) using third-generation selfed progeny (estimated homozygosity rate = 0.96). Young leaf tissue was used to extract high molecular weight DNA (> 50kb) for HiFi PacBio long-read sequencing and to construct an Hi-C/Omni-C library for Illumina short-read sequencing. The 29× and 26× genome coverage of HiFi and Illumina reads, respectively, for de novo assembly yielded 2,651 contigs (9.1 Gbp, N50 contig size 12.0 Mbp). Hi-C analysis mapped 97% of the nucleotides on 11 chromosomes. The assembly was verified through comparison with a consensus linkage map comprising 7,781 markers. BUSCO analysis identified ∼ 91% conserved genes. Annotations of genes and comparisons of repeat elements with other Cupressaceae and Pinaceae species provide a fundamental resource for conifer research.

Comparison of Four Different DNA Isolation Methods from MGIT Culture for Long-Read Whole Genome Sequencing of Mycobacterium tuberculosis

Background: Tuberculosis (TB) remains a global health challenge, particularly due to drug resistance and limitations in rapid diagnosis. Next-generation sequencing (NGS), especially long-read whole genome sequencing (WGS), shows promise for rapidly detecting TB and drug resistance, but it requires high-quality DNA, which is difficult to extract from Mycobacterium tuberculosis due to its complex cell wall. Objectives: This study evaluated four DNA isolation methods for extracting pure DNA from M. tuberculosis, aiming to standardize protocols for long-read WGS. Methods: Mycobacterium tuberculosis H37RV colonies were grown in BACTEC MGIT liquid medium. Two pellets were prepared as the initial material for the DNA extraction protocol: Pellets from 1 mL McFarland 2 suspensions and all growing colonies from two MGIT liquid cultures. Four DNA extraction methods were used: The cetyltrimethylammonium bromide (CTAB) method, GeneJET Genomic DNA Purification Kit, Quick-DNA Fecal/Soil Microbe Kit, and Genematrix Tissue/Bacterial DNA Purification Kit, with some modifications. DNA quality was assessed based on concentration, purity, and integrity. Results: Among the tested methods, the Quick-DNA Fecal/Soil Kit yielded approximately 85 ng/mL of DNA and a purity of 1.9 at 260/280 nm from the colonial pellet of two MGIT tubes. However, lower intact DNA [DNA integrity number (DIN) ~ 6.8] was obtained with this kit. The CTAB method provided the highest intact DNA (DIN ~ 9.5), although the purity of the DNA was not sufficient. Conclusions: Based on three repetitions of McF-2 and colonial pellet extractions, the Quick-DNA Fecal/Soil Kit yielded the highest DNA quantity and purity but showed lower integrity compared to other methods, indicating the need for adjustments. A pellet from two MGIT cultures (~ 100 µL) is suitable for long-read WGS with this kit. However, a larger sample size is required to generalize these findings. For effective long-read sequencing of M. tuberculosis, DNA extraction protocols must be optimized to balance yield, fragment size, and purity for accurate sequencing and drug resistance analysis.

DmTGS: Precise Targeted Enrichment Long-Read Sequencing Panel for Tandem Repeat Detection.

Tandem repeats (TRs) are abundant in the human genome and associated with repeat expansion disorders. Our study aimed to develop a tandem repeat panel utilizing targeted long-read sequencing to evaluate known TRs associated with these disorders and assess its clinical utility. We developed a targeted long-read sequencing panel for 70 TR loci, termed dynamic mutation third-generation sequencing (dmTGS), using the PacBio Sequel II platform. We tested 108 samples with suspected repeat expansion disorders and compared the results with conventional molecular methods. For 108 samples, dmTGS achieved an average of 8000 high-fidelity reads per sample, with a mean read length of 4.7 kb and read quality of 99.9%. dmTGS outperformed repeat-primed-PCR and fluorescence amplicon length analysis-PCR in distinguishing expanded from normal alleles and accurately quantifying repeat counts. The method demonstrated high concordance with confirmatory methods (rlinear = 0.991, P < 0.01), and detected mosaicism with sensitivities of 1% for FMR1 CGG premutation and 5% for full mutations. dmTGS successfully identified interruptive motifs in genes that conventional methods had missed. For variable number TRs in the PLIN4 gene, dmTGS identified precise repeat counts and sequence motifs. Screening 57 patients with suspected genetic muscular diseases, dmTGS confirmed repeat expansions in genes such as GIPC1, NOTCH2NLC, NUTM2B-AS1/LOC642361, and DMPK. Additionally, dmTGS detected CCG interruptions in CTG repeats in 8 myotonic dystrophy type 1 patients with detailed characterization. dmTGS accurately detects repeat sizes and interruption motifs associated with repeat expansion disorders and demonstrates superior performance compared to conventional molecular methods.

Fully phased genome assemblies and graph-based genetic variants of the olive flounder, Paralichthys olivaceus

The olive flounder, Paralichthys olivaceus, also known as the Korean halibut, is an economically important flatfish in East Asian countries. Here, we provided four fully phased genome assemblies of two different olive flounder individuals using high-fidelity long-read sequencing and their parental short-read sequencing data. We obtained 42–44 Gb of ~15-kb and ~Q30 high-fidelity long reads, and their assembly quality values were ~53. We annotated ~30 K genes, ~170-Mb repetitive sequences, and ~3 M 5-methylcytosine positions for each genome assembly, and established a graph-based draft pan-genome of the olive flounder. We identified 5 M single-nucleotide variants and 100 K structural variants with their genotype information, where ~13% of the variants were possibly fixed in the two Korean individuals. Based on our chromosome-level genome assembly, we also explored chromosome evolution in the Pleuronectiformes family, as reported earlier. Our high-quality genomic resources will contribute to future genomic selection for accelerating the breeding process of the olive flounder.

MAGqual: a stand-alone pipeline to assess the quality of metagenome-assembled genomes

BackgroundMetagenomics, the whole genome sequencing of microbial communities, has provided insight into complex ecosystems. It has facilitated the discovery of novel microorganisms, explained community interactions and found applications in various fields. Advances in high-throughput and third-generation sequencing technologies have further fuelled its popularity. Nevertheless, managing the vast data produced and addressing variable dataset quality remain ongoing challenges. Another challenge arises from the number of assembly and binning strategies used across studies. Comparing datasets and analysis tools is complex as it requires the quantitative assessment of metagenome quality. The inherent limitations of metagenomic sequencing, which often involves sequencing complex communities, mean community members are challenging to interrogate with traditional culturing methods leading to many lacking reference sequences. MIMAG standards aim to provide a method to assess metagenome quality for comparison but have not been widely adopted.ResultsTo address the need for simple and quick metagenome quality assignation, here we introduce the pipeline MAGqual (Metagenome-Assembled Genome qualifier) and demonstrate its effectiveness at determining metagenomic dataset quality in the context of the MIMAG standards.ConclusionsThe MAGqual pipeline offers an accessible way to evaluate metagenome quality and generate metadata on a large scale. MAGqual is built in Snakemake to ensure readability and scalability, and its open-source nature promotes accessibility, community development, and ease of updates. MAGqual is built in Snakemake, R, and Python and is available under the MIT license on GitHub at https://github.com/ac1513/MAGqual.EB_9RbKd3CYdEq3zCTHKaqVideo

Genetic Variation in Jamaican Populations of the Coffee Berry Borer, Hypothenemus hampei.

The coffee berry borer (CBB) Hypothenemus hampei was first described in Africa in 1867 and has spread to all major coffee-producing regions worldwide, including Jamaica. Using long-read sequencing, we produced a new high-quality reference genome (172.7 Mb) for the Jamaican strain of the CBB, with 93% of the genome assembled into 14 scaffolds. Whole genome sequencing of pooled samples from different populations across Jamaica showed that the CBB harbors low levels of genetic diversity alongside an excess of low-frequency alleles, indicative of a recent genetic bottleneck. The analyses also showed a recent surge in the activity of transposable elements (TEs), particularly LINE/R1 and LTR/Gypsy elements, within CBB populations. Our findings offer first insights into the evolutionary genomics of CBB populations in Jamaica, highlighting the potential role of TEs in shaping the genome of this important pest species.

The chromosome-level Elaeagnus mollis genome and transcriptomes provide insights into genome evolution, glycerolipid and vitamin E biosynthesis in seeds

Elaeagnus mollis, which has seeds with high lipid and vitamin E contents, is a valuable woody oil plant with potential for utilization. Currently, the biosynthesis and regulation mechanism of glycerolipids and vitamin E are still unknown in E. mollis. Here, we present the chromosome-level reference genome of E. mollis (scaffold N50: ~40.66Mbp, genome size: ~591.48Mbp) by integrating short-read, long-read, and Hi-C sequencing platforms. A total of 36,796 protein-coding sequences, mainly located on 14 proto-chromosomes, were predicted. Additionally, two whole genome duplication (WGD) events were suggested to have occurred ~54.07 and ~35.06 million years ago (MYA), with Elaeagnaceae plants probably experiencing both WGD events. Furthermore, the long terminal retrotransposons in E. mollis were active ~0.23MYA, and one of them was inferred to insert into coding sequence of the negative regulatory lipid synthesis gene, EMF2. Through transcriptomic and metabonomic analysis, key genes contributing to the high lipid and vitamin E levels of E. mollis seeds were identified, while miRNA regulation was also considered. This comprehensive work on the E. mollis genome not only provides a solid theoretical foundation and experimental basis for the efficient utilization of seed lipids and vitamin E, but also contributes to the exploration of new genetic resources.

Leveraging the T2T assembly to resolve rare and pathogenic inversions in reference genome gaps.

Chromosomal inversions (INVs) are particularly challenging to detect due to their copy-number neutral state and association with repetitive regions. Inversions represent about 1/20 of all balanced structural chromosome aberrations and can lead to disease by gene disruption or altering regulatory regions of dosage-sensitive genes in cis Short-read genome sequencing (srGS) can only resolve ∼70% of cytogenetically visible inversions referred to clinical diagnostic laboratories, likely due to breakpoints in repetitive regions. Here, we study 12 inversions by long-read genome sequencing (lrGS) (n = 9) or srGS (n = 3) and resolve nine of them. In four cases, the inversion breakpoint region was missing from at least one of the human reference genomes (GRCh37, GRCh38, T2T-CHM13) and a reference agnostic analysis was needed. One of these cases, an INV9 mappable only in de novo assembled lrGS data using T2T-CHM13 disrupts EHMT1 consistent with a Mendelian diagnosis (Kleefstra syndrome 1; MIM#610253). Next, by pairwise comparison between T2T-CHM13, GRCh37, and GRCh38, as well as the chimpanzee and bonobo, we show that hundreds of megabases of sequence are missing from at least one human reference, highlighting that primate genomes contribute to genomic diversity. Aligning population genomic data to these regions indicated that these regions are variable between individuals. Our analysis emphasizes that T2T-CHM13 is necessary to maximize the value of lrGS for optimal inversion detection in clinical diagnostics. These results highlight the importance of leveraging diverse and comprehensive reference genomes to resolve unsolved molecular cases in rare diseases.

From acute to persistent infection: revealing phylogenomic variations in Salmonella Agona.

Salmonella enterica serovar Agona (S. Agona) has been increasingly recognised as a prominent cause of gastroenteritis. This serovar is a strong biofilm former that can undergo genome rearrangement and enter a viable but non-culturable state whilst remaining metabolically active. Similar strategies are employed by S. Typhi, the cause of typhoid fever, during human infection, which are believed to assist with the transition from acute infection to chronic carriage. Here we report S. Agona's ability to persist in people and examine factors that might be contributing to chronic carriage. A review of 2233 S. Agona isolates from UK infections (2004-2020) and associated carriage was undertaken, in which 1155 had short-read sequencing data available. A subset of 207 isolates was selected from different stages of acute and persistent infections within individual patients. The subset underwent long-read sequencing and genome structure (GS) analysis, as well as phenotyping assays including carbon source utilisation and biofilm formation. Associations between genotypes and phenotypes were investigated to compare acute infections to those which progress to chronic. GS analysis revealed the conserved arrangement GS1.0 in 195 isolates, and 8 additional GSs in 12 isolates. These rearranged isolates were typically associated with early, convalescent carriage (3 weeks- 3 months). We also identified an increase in SNP variation during this period of infection. We believe this increase in genome-scale and SNP variation reflects a population expansion after acute S. Agona infection, potentially reflecting an immune evasion mechanism which enables persistent infection to become established.

A novel approach to detecting microduplication in split hand/foot malformation type 3 at the single-cell level: SHFM as a case study

BackgroundSplit hand/foot malformation (SHFM) is a congenital limb deficiency characterized by missing or shortened central digits. Several gene loci have been associated with SHFM. Identifying microduplications at the single-cell level is challenging in clinical practice, and traditional detection methods may lead to misdiagnoses in embryos and pregnant women.ResultsIn this research, we utilized a low cell count and whole-genome amplification products to employ single nucleotide polymorphism arrays, next-generation sequencing, and third-generation sequencing methods to detect copy number variants of microduplications in a SHFM3 case with limited DNA. Additionally, Karyomapping and combined linkage analysis were conducted to validate the results.ConclusionsThis study establishes a new strategy for identifying microduplications or microdeletions at the single-cell level in clinical preimplantation genetic testing, enhancing the efficiency and accuracy of diagnosing microduplication or microdeletion diseases during IVF-PGT and prenatal diagnosis.

Genomic characterization of a clonal emergent Salmonella Minnesota lineage in Brazil reveals the presence of a novel megaplasmid of resistance and virulence.

Salmonella Minnesota has emerged in Brazil as the predominant serovar in poultry and poultry products, along with Salmonella Heidelberg. To understand the emergence of Salmonella Minnesota over the last few years in Brazil, we performed a comparative analysis between 69 selected S. Minnesota genomes from Pathogen Detection database and 65 clonal emergent genomes isolated from Brazil. We demonstrate the presence of multidrug resistance genes against tetracycline [tet(A)], sulfonamide (sul2), and AmpC beta-lactamase (blaCMY-2) in emergent genomes, along with the carriage of a megaplasmid of resistance and virulence (~210 kb), designated pESM (plasmid for emergent Salmonella Minnesota). pESM is an IncC/A2 plasmid predicted to increase S. Minnesota environmental tolerance to mercury (mer operon) and provide resistance to tetracycline and ampicillin due to the presence of tet(A) and blaCMY-2, respectively. Moreover, pESM carries the yersiniabactin siderophore (high-pathogenicity island of Yersinia) related to the iron uptake. The temporal inference demonstrated that the most recent common ancestor dated from ~1978 and that the clonal emergent genomes carrying the pESM belong to a completely different lineage of S. Minnesota. Our results indicate that the presence of pESM likely contributes to the emergence of S. Minnesota and is precisely related to the successful spread of this particular clonal lineage in Brazil.IMPORTANCESalmonella Minnesota has emerged in Brazil as one of the leading serovars related to human and animal infection, presenting high virulence and antibiotic resistance to drugs classified as the highest priority for clinical treatment in humans. This study performed whole-genome sequencing, temporal analysis, and phylogenetics to understand the genetic insights related to the emergence of Salmonella Minnesota in Brazil. Long-read sequencing has led to the identification and characterization of a unique megaplasmid carrying virulence, antibiotic resistance, and heavy-metal tolerance genes, which may play a central role in S. Minnesota's successful emergence in Brazil and possibly worldwide. The potentially high transmissibility of this plasmid between clones and serovars represents a risk to public health since its acquisition may increase Salmonella's fitness, virulence, resistance, and persistence. Understanding the genetic aspects related to the emergence of serovars can help devise measures to mitigate the spread of hazardous multidrug-resistant strains.

Chromosomal-level genome assembly of the Antarctic sea urchin Sterechinus neumayeri, a model for Antarctic invertebrate biology.

The Antarctic sea urchin Sterechinus neumayeri (Echinoida;Echinidae) is routinely used as a model organism for Antarctic biology. Here, we present a high-quality genome of S. neumayeri. This chromosomal-level assembly was generated using PacBio long-read sequencing and HiC chromatin conformation capture sequencing. This 885.3 Mb assembly exhibits high contiguity with a scaffold length N50 of 36.7Mb assembled into 20 chromosomal length scaffolds. These putative chromosomes exhibit a high degree of synteny compared to other sea urchin models. We used transcript evidence gene modeling combined with sequence homology to identify 21,638 gene models that capture 97.4% of BUSCO orthologs. Among these, we were able to identify and annotate conserved developmental gene regulatory network orthologs, positioning S. neumayeri as a tractable model for comparative studies on evolution and development.

Long-read RNA sequencing reveals allele-specific N6-methyladenosine modifications.

Long-read sequencing technology enables highly accurate detection of allele-specific RNA expression, providing insights into the effects of genetic variation on splicing and RNA abundance. Furthermore, the ability to directly sequence RNA using the Oxford Nanopore technology promises the detection of RNA modifications in tandem with ascertaining the allelic origin of each molecule. Here, we leverage these advantages to determine allele-biased patterns of N6-methyladenosine (m6A) modifications in native mRNA. We utilized human and mouse cells with known genetic variants to assign allelic origin of each mRNA molecule combined with a supervised machine learning model to detect read-level m6A modification ratios. Our analyses revealed the importance of sequences adjacent to the DRACH-motif in determining m6A deposition, in addition to allelic differences that directly alter the motif. Moreover, we discovered allele-specific m6A modification (ASM) events with no genetic variants in close proximity to the differentially modified nucleotide, demonstrating the unique advantage of using long reads and surpassing the capabilities of antibody-based short-read approaches. This technological advancement promises to advance our understanding of the role of genetics in determining mRNA modifications.