Reference Genome Research Articles

In forensic genetics, Y-chromosomal short tandem repeats (Y-STRs) are widely used in male lineage tracing, kinship analysis, and mixture interpretation. Conventional genotyping of Y-STRs relies on length-based detection via capillary electrophoresis (CE). However, massively parallel sequencing (MPS) enables simultaneous determination of both length and sequence genotypes, thereby increasing the information obtained per assay. While developing and validating an MPS panel, we made an unexpected observation: the DYS572 locus exhibited heterozygous genotypes in both male and female samples. This could misidentify a single-source sample as a mixed sample. Reference sequence alignment revealed that the DYS572 region on the Y chromosome (GRCh38, chrY: 3,811,419-3,811,858) shares 98.41% sequence identity (432/439bp) with a homologous region on the X chromosome (GRCh38, chrX: 90,242,118-90242557). The corresponding X-chromosomal region harbors seven single nucleotide variants (SNVs) distinguishing it from the Y sequence: four upstream of the repeat region at positions - 183 (rs1031074259), - 62 (rs34553462), - 56 (rs3959844), and - 47 (rs36022122), and three downstream at positions + 6 (rs35114058), + 96 (rs2752369), and + 194 (rs2752370). In practical applications, if primer design or bioinformatic filtering is suboptimal, amplification products of DYS572 may originate from the X chromosome. This study provides a thorough characterization of the DYS572 locus on the sex chromosomes, along with the genotyping patterns observed in males and females. This information will be valuable for future forensic applications.

Sweetpotato (Ipomoea batatas L. Lam) production is threatened by complex viral diseases, notably sweet potato virus disease (SPVD) worldwide, which results from co-infection by sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV). This study provides virus-specific transcriptomic insights into the immune responses of three sweetpotato cultivars, ‘Beauregard’, ‘Tanzania’, and ‘New Kawogo’, to SPFMV, SPCSV, and SPVD. Using RNA-seq profiling across three timepoints post-infection at 3, 6, and 12 weeks, we identified distinct virus- and genotype-specific gene expression responses. ‘New Kawogo’ activated early and sustained immune pathways involving redox regulation, transcriptional control, and hormonal signaling in response to both SPCSV and SPFMV, while showing minimal transcriptional disruption under SPVD, reflecting robust tolerance. ‘Beauregard’ exhibited early suppression of immune and metabolic genes, with delayed and disorganized recovery efforts, particularly under SPVD. Defense-related pathways including NBS-LRR signaling, RNA silencing, and hormonal regulation were consistently upregulated in ‘New Kawogo’ and to a lesser extent in ‘Tanzania’, but remained inactive in ‘Beauregard’. This study highlights candidate resistance and susceptibility genes for each virus, providing a molecular basis for developing virus-resilient sweetpotato cultivars through functional genomics and marker-assisted breeding. These findings elucidate the molecular basis of virus resistance in sweetpotato and identify candidate genes for marker-assisted breeding, despite limitations arising from the use of a diploid reference genome and discrete sampling intervals.

Copper is essential for all living organisms, but becomes toxic when present in excess. Biological systems have evolved mechanisms to keep organisms in copper homeostasis. Studies have shown that Salmonella can acquire mobile genetic elements that provide enhanced tolerance to stressed environments, such as the Copper Homeostasis and Silver Resistance Island (CHASRI), which has become more prevalent in certain serovars that are exposed to higher levels of copper. In this study, whole genome sequence data available from NCBI Pathogen Detection was used to determine the incidence of the CHASRI is in Salmonella enterica isolates. The results show that the CHASRI is present in a wider range of serovars than previously known and can be found in isolates from different food sources, including nuts, spices, and produce. Salmonella Genomic Island-4 (SGI-4) was previously described as the primary mobile element through which the CHASRI was transferred to Salmonella; however, results from this comparative study of closed reference genomes identified additional integrations of the CHASRI as both a singular mobile element and as a component of an SGI-4 variant.

This study aims to characterize tissue-specific expression patterns in Hanwoo steers by identifying co-expression modules, functional pathways, and hub genes related to fat and muscle traits using Weighted Gene Co-expression Network analysis (WGCNA). RNA-Seq data were generated from three muscle tissues (longissimus muscle, tenderloin, and rump) and two fat tissues (back fat and abdominal fat) collected from six 30-month-old Hanwoo steers. Quality control of raw sequencing reads was performed using FastQC, and trimmed reads were aligned to the bovine reference genome (ARS-UCD1.3) using HISAT2. We also identified a gene co-expression network via WGCNA using normalized gene expression values. Modules were defined based on topological overlap and correlated with tissue-specific expression patterns. Modules with a significant association (p < 0.05) were used for functional enrichment based on Gene Ontology (GO) and KEGG pathways, as well as Protein–Protein Interaction Network analysis. A total of seven co-expression modules were identified by WGCNA and labeled in distinct colors (yellow, blue, red, brown, turquoise, green, black). Among them, the yellow and blue modules were positively associated with back fat, while the turquoise and green modules showed a negative correlation with abdominal fat. Additionally, the turquoise or green module was positively correlated with longissimus and rump tissues, indicating distinct gene expression patterns between fat and muscle. This study identified key co-expression modules and hub genes associated with muscle and fat metabolism. Notably, ARPC5 (blue module) was involved in lipid metabolism and energy storage, whereas AGPAT5 (turquoise module) was linked to maintaining muscle cell structure and function. These findings reveal biological mechanisms for tissue-specific gene regulation, providing targets for enhancing meat quality in Hanwoo.

Stream-dwelling fishes face diverse hydrological pressures, making the broadly distributed Opsariichthys bidens an ideal model for analyzing adaptive evolution. To elucidate its adaptation to a high-dissolved-oxygen and high-flow-velocity stream environment, a high-quality genome with comprehensive annotation is essential. In this study, we present the first telomere-to-telomere (T2T) reference genome for O. bidens, constructed using PacBio HiFi, Oxford Nanopore Ultra-long, and Hi-C technologies. The assembled genome spans 841.96 Mb, comprising 38 chromosomes, each in a single contig (contig N50 = 22.42 Mb, 2.5-fold higher than the previous version), achieving a gap-free standard with 99.34% BUSCO completeness. Additionally, 38 centromeric sequences, 37 double-telomeric sequences, and 1 single-telomeric sequence were successfully identified, providing essential molecular markers. Phylogenetic analysis revealed a divergence time of 13.5 million years between O. bidens and its closely related species Z. platypus, with collinearity analysis confirming their high genomic conservation. Gene family analysis revealed 350 expanded families enriched in pathways associated with adaptation to high-dissolved-oxygen environments (e.g., antioxidant defense, oxidative phosphorylation, mitochondrial electron transport chain) and high-flow-velocity environments (e.g., exercise endurance, myocardial contraction, actin binding). Positive selection analysis further identified multiple pathways and key genes involved in mitochondrial optimization, oxygen utilization, and metabolic regulation. The T2T assembly greatly improves assembly continuity and enabling precise identification of centromeres and telomeres for O. bidens. These results provide a robust foundation for studying its adaptive evolution to stream environment.

BackgroundAmaranthus tuberculatus (waterhemp) is a troublesome agronomic weed species that is dioecious with an XY sex-determination system. The evolution of the sex-determining region (SDR), the contiguity of the region, genomic landscape, and the expression pattern of genes within the region remain poorly understood.ResultsWe assembled a high-quality, chromosome-level nuclear genome and chloroplast and mitochondrial genomes of a male A. tuberculatus. Combining the genomes with restriction site-associated DNA genome-wide association (RAD-GWA) analysis, comparative genomics, adaptive evolution analysis, and transcriptomic profiling, we identified a ~ 31.8 Mb region on chromosome 1 that is strongly associated with sex. This region is gene-poor, abundant in long terminal repeat (LTR) retrotransposons, and harbors two inversions and a 3.19 Mb haplotype-specific region. Synteny analysis revealed that chromosome 1 likely originated from the fusion of two ancestral chromosomes, and mRNA analysis indicated 76 genes out of the 528 protein-coding genes within the putative SDR of Hap1 were differentially expressed between mature male and female flowers, with several of the genes enriched for Gene Ontology (GO) terms involved in floral development. We further characterized the phosphatidylethanolamine binding protein (PEBP) family in A. tuberculatus and related species to gain insights into FLOWERING LOCUS T diversity, as well as identified nuclear insertions of organellar origin in the species.ConclusionOur results provide insight into the evolution of a sex-associated region in a weedy dioecious species, and the diversity of the PEBP protein family in amaranths. The genomic resources from this study will also be valuable for addressing further questions on adaptive trait evolution within the genus as well as questions surrounding dioecy in this and other plant species.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12181-w.

Abstract This study documents the recent occurrence of Sophonia orientalis (Hemiptera: Cicadellidae) on avocado ( Persea americana ) crops in the Gharb and Loukkos regions of northwest Morocco. Species identification was validated through morphological examination of male genitalia and DNA analysis using standard barcoding techniques . The Two spotted leafhopper S. orientalis is a polyphagous and invasive pest native to Southeast Asia, and it has been recorded in various locations around the world. In Morocco, the area planted with avocados is constantly increasing due to the crop's profitability. However, the emergence of leafhoppers poses significant phytosanitary threats to growers. Identification based on male genitalia features and confirmation by DNA sequencing revealed infestations of S. orientalis , with high sequence identity (99.11%–100%) to reference sequences in GenBank. Field surveys from March to September 2024 showed that S. orientalis infestations were mostly found in areas between Moulay Bousselham and Sidi Allal Tazi, with up to 50% of the inspected avocado orchards indicating its establishment in those regions. Although no crop losses have yet been reported in Morocco due to S. orientalis , its confirmed establishment in a significant proportion of avocado orchards and its invasive nature in other regions justify the early implementation of integrated pest management (IPM) strategies. Coordinated efforts among agricultural authorities, researchers, and farmers are essential to safeguard Morocco's avocado industry while maintaining sustainable agricultural practices.

Northern corn leaf blight is a major fungal disease hindering maize production worldwide. Among the various strategies of disease management, the deployment of host plant resistance is the most economic means to mitigate the yield losses, as it is cost-effective and durable. In this study, we performed the genome-wide association study (GWAS) analysis in a set of 336 maize inbred lines. The experimental material was evaluated for northern corn leaf blight disease response across two seasons during the rainy seasons of 2023 and 2024. The ANOVA results and estimates of genetic variability parameters indicated the existence of a substantial amount of genetic variability. High heritability and high genetic advance as percent mean suggested the presence of additive genetic effects in controlling the disease response. GWAS analysis was performed employing GLM, MLM, CMLM, MLMM, FarmCPU and BLINK. The results from GWAS identified 74 marker associations from GLM and FarmCPU models. The QTN S1_7356398, located on chromosome 1, identified from the GLM model, explained 12.12 percent of phenotypic variation. Another QTN S2_51098833 located on chromosome 2, identified from the FarmCPU model, explained 6.14 percent variation. Remaining associations explained lesser PVE, suggesting the quantitative inheritance of NCLB resistance. Candidate gene identification was performed by keeping B73 as a reference genome. The identified QTNs from the current study were found to be located in annotated genes with functional domains implicated in defence mechanisms in maize and other crops. Many candidate genes, including chitinase, putative serine/threonine protein kinase, and aldehyde oxygenase, were identified and found to play a crucial role in plant defence mechanisms against several biotic and abiotic stresses.

Myrothamnus flabellifolia is a dioecious resurrection plant endemic to southern Africa that has become an important model for understanding desiccation tolerance. Despite its ecological and medicinal significance, genomic and transcriptomic resources for the species are limited. We generated a chromosome-level, haplotype-resolved reference genome assembly and annotation for M. flabellifolia and conducted transcriptomic profiling across a natural dehydration-rehydration time course in the field. Genome architecture and sex determination were characterized, and co-expression network and cis-regulatory element (CRE) enrichment analyses were used to investigate dynamic responses to desiccation. The 1.28-Gb genome exhibits unusually consistent chromatin architecture with unique chromosome organization across highly divergent haplotypes. We identified an XY sexual system with a small sex-determining region on Chromosome 8. Transcriptomic responses varied with dehydration severity, pointing to early suppression of growth, progressive activation of protective mechanisms, and subsequent return to homeostasis upon rehydration. Late embryogenesis abundant and early light-induced protein transcripts were dynamically regulated and showed enrichment of abscisic acid and stress-responsive CREs pointing toward conserved responses. Together, this study provides foundational resources for understanding the genomic architecture and reproductive biology of M. flabellifolia and offers new insights into the mechanisms of desiccation tolerance.

Soybean (Glycine max L.) is widely cultivated in northeastern Italy, especially in Friuli-Venezia Giulia and Veneto. Since 2013, during early seedling development, necrotic lesions have appeared on upper leaves, spreading downward and causing up to 25% defoliation in the field. Purplish discoloration was observed on petioles, stems, and pods. Seeds showed pale to dark purple blotches on the seed coat, from small spots to full coverage. These symptoms are typical of Cercospora leaf blight (CLB) and Purple Seed Stain (PSS). In 2018-2019, symptomatic tissues were analyzed. Small fragments of infected stems and pods from five different fields (ten plants/site), were surface-sterilized in 1% sodium hypochlorite for 10 min, rinsed with sterile distilled water, and plated onto Potato Dextrose Agar (BD Difco™) with streptomycin sulphate (300 mg/L). Plates were incubated in the dark at 25°C for 7 days. Seeds were disinfected similarly, but for 1 minute. Single-spore cultures produced deep purple mycelium on the lower surface and grey on the upper. Stromata were absent or poorly developed. Conidiophores were straight, pale to dark brown, 120–180 × 3.0–5.5 µm; conidia were solitary, hyaline, slightly curved, and measured 40–250 × 2.5–4.5 µm (n=25). Eight representative isolates from Friuli-Venezia Giulia, three from pods (CREA-CI24, CREA-CI25, CREA-CI26) and five from seeds (C-N7, C-170-1, C-203-4, C-203-5, C-212), underwent multilocus molecular analysis (Groenewald et al., 2012). DNA was extracted and internal transcribed spacer region (ITS), calmodulin (CAL), and histone H3 (HIS3) were amplified (White et al., 1990; Carbone and Kohn, 1999; Crous et al., 2004) and Sanger sequenced. Sequences were deposited in GenBank (ITS: MK989496–98, PX092092–96; CAL: MK991296–98, PX091946–50; HIS3: MK991293–95, PX091951–55). MegaBLAST analysis showed 99% to 100% identity with reference sequences of C. cf. flagellaris (ITS: JX143614, CAL: JX142867, HIS3: JX142621). Phylogenetic analysis using concatenated sequences and 63 references confirmed clustering within C. cf. flagellaris. Pathogenicity tests (Cai & Schneider, 2005) were conducted with strains CI24, CI25, and CI26, in triplicate. Sterile soybean leaves on living plants were sprayed with conidial suspension (10⁴ conidia/mL) and incubated at 25°C under moist conditions. After 8–10 days, necrosis and blight symptoms developed as in the field. No symptoms were seen in controls. Reisolation from symptomatic inoculated leaves confirmed C. cf. flagellaris as the causal agent, based on morphology and ITS sequences. The symptoms observed in this study align with Cercospora-related diseases (CLB and PSS). Previously attributed to C. kikuchii, recent studies (Soares et al., 2015; Albu et al., 2016) showed that C. cf. flagellaris and C. cf. sigesbeckia are also involved. Fernandes et al. (2025) reported that C. cf. flagellaris causes milder symptoms on young soybean leaves than other Cercospora spp., consistent with our observations. To our knowledge, this is the first report of C. cf. flagellaris on soybean in Italy. Soybean covers about 300,000 ha in the country, mainly in northeastern valleys, and is a key food and feed crop. This finding expands known Cercospora diversity associated with CLB and PSS in Italy and highlights the need for further studies on pathogen distribution, epidemiology, cultivar susceptibility, and management strategies to sustain soybean production.

Big Basin Sagebrush (Artemisia tridentata ssp. tridentata) is a common shrub found across high desert and arid regions of North America. Although its natural distribution has been reduced over the past century due to farm and urban expansion it remains a keystone species in much of the high desert and is a host for native arthropod species. However, there have been few studies that examined the effect of phytopathogens on A. tridentata (Allen & West 1987; Welch & Nelson 1995). During a survey for alternate hosts of the X-disease phytoplasma (‘Candidatus Phytoplasma pruni’) (Shires et al. 2025) we observed phytoplasma-like foliar chlorosis and dieback-like symptoms on A. tridentata in uncultivated areas of Chelan County, WA. Approximately 30-40% of plants were symptomatic at these sites, and symptom progression was seasonal, with the emergence of enlarged, pale-yellow chlorotic leaves between late spring (May-June) and summer (July-August). Chlorosis began at the leaf apex and extended towards the petiole, with tip necrosis then leaf drop occurring in late August (Figure 1). Dieback of shoot apices and limbs was also observed on individual plants. Leaf and stem tissues were collected from 19 symptomatic and 63 asymptomatic sagebrush at three sites in Chelan Co., DNA extracted using the Qiagen DNeasy plant mini kit (Qiagen Corp. Germantown, MD) per the manufacturer’s instructions, and tested for 16SrIII phytoplasmas on the basis of proximity to orchards known to be infected with ‘Ca. P. pruni’ by qPCR as per Shires et al. (2025). All symptomatic plants tested positive, while asymptomatic plants did not. For identification, the partial 16S rRNA gene was amplified from seven samples by nested PCR using the P1/P7 (Deng & Hiruki 1991) then R16F2n/R2 primers (Gundersen & Lee 1996); resulting products were Sanger-sequenced (Eton Biosciences, San Diego, CA) and deposited in GenBank (PV994682-PV994688). Examination using iPhyClassifier (Zhao et al. 2012) indicated that these were a ‘Ca. P. pruni’-related strain with 99.66% identity to reference sequence JQ0443393. BLASTn indicated a 100% nucleotide identity match to Lilac decline phytoplasma strain LlcDec1 (KP877578) (Davis et al. 1996). For further identification, the partial secY (849 bp), secA (490 bp), Ef-Tu (793 bp), and imp (457 bp) genes were amplified and sequenced as per Molnar et al. (2024). Of these secY (PX023655-PX023661), secA (PX023662-PX023668) and Ef-Tu (PX023669-PX023675) had 99.8%, 98.9% and 98.5% identity to X-disease strains PX11CT1 (JQ268254), 20-15 (OR67122), and 22-2470 (OR670177) respectively, whereas imp (PX023676-PX023682) had 96.9 % identity to PoIBI strain “Annette Hegg Maxi” (AB636364) (100% coverage for all fragments). Neighbor-joining phylogenetic analysis of the concatenated marker regions showed that this phytoplasma forms a separate clade from other 16SrIII strains, and whose closest relative is PoiBI strain JR1 (ASM30946v1) with 98.9% identity across the aligned region (Figure 2). Cumulatively these data suggest the Sagebrush yellows-associated phytoplasma is a ‘Ca. P. pruni’-related strain that is novel and distinct from previously described members of the 16SrIII group, based on sequence identity and the host on which it causes disease. This find is significant as it is the first report of a novel phytoplasma infecting a keystone species in western North America and an indicator of where 16SrIII phytoplasmas infecting cultivated plants may have originated.

Background/Objectives: According to the World Drug Report 2025, cannabis is the most abused drug in the world, being sold in illicit markets in various physical forms ranging from herbal cannabis to cannabis resin and liquid cannabis. Currently, the methods used for cannabis identification are largely based on the morphological features and chemical content of the product. In this respect, identification could be severely impacted if the product is highly fragmented or pulverised. As such, DNA-based molecular techniques offer a viable alternative detection approach. In this study, we have developed a robust DNA testing method for cannabis identification, with high sensitivity and specificity. Methods/Results: Two plant DNA barcode regions, rbcL and matK, were successfully amplified in a cohort of 54 cannabis plant samples. DNA sequences obtained from these samples were blast-searched against GenBank and resulted in returned matched identity of at least 99% compared to their corresponding Cannabis sativa reference sequences. In addition, the amplification of two cannabis-unique markers, the tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes, produced amplicons with expected sizes only in cannabis samples; these amplicons were not detected in those plants closely related to cannabis. Sequence comparison of the majority of samples yielded at least 97% matched identity against C. sativa reference sequences in GenBank. The THCAS and CBDAS markers detected only the cannabis DNA in varying levels of cannabis–hops and cannabis–tobacco DNA mixtures. Lastly, the use of the four markers could effectively differentiate between cannabis and non-cannabis in 27 blinded samples, including 18 actual casework samples. Conclusions: In conclusion, these four genetic markers can be used to discriminate cannabis from other plant species at the genus level, especially in challenging forensic samples lacking morphological features which therefore cannot be determined by traditional detection methods. As such, this method can complement existing techniques to identify a myriad of cannabis samples.

Development of a double-probe rolling circle amplification-lateral flow dipstick assay for rapid detection of Amphidinium carterae.

A novel glycerophosphodiester phosphodiesterase 13 is involved in the phosphate starvation-induced phospholipid degradation in rice.

Molecular evidence of Histomonas meleagridis in Ascaridia galli from chickens in Thailand: Possibility of transmission pathways.

Butuo Black sheep (BBS), an ancient indigenous Chinese breed, has co-evolved with the Yi people's semi-nomadic lifestyle and demonstrated exceptional adaptability to high-altitude migrations. However, a high-quality reference genome for BBS is still lacking. In this study, we established a high-quality chromosome-level genome assembly of BBS using PacBio HiFi sequencing. The final assembled genome size was approximately 2.95 Gb, with a contig N50 of 71.45 Mb and a scaffold N50 of 92.26 Mb. The genome assembly achieved a high Benchmarking Universal Single-Copy Orthologs (BUSCO) score of 95.9%, indicating its high completeness and quality. The de novo genome prediction revealed that repetitive sequences accounted for 47.74% of the genome, with long interspersed nuclear elements (LINEs) being the most abundant. Additionally, we present 50 BBS shotgun genomes sequenced using the Illumina HiSeq 2000 platform, with a mean coverage of 10.36×. The study generated approximately 1.2 terabytes of raw data, with 99.9% clean reads mapping successfully to the sheep reference genome at 99.6% coverage. This extensive dataset provides a valuable resource for studying genetic diversity and evolutionary patterns in BBS.

Albizia odoratissima is a valuable drought-tolerant native tree species in the dry and hot river valleys of China, which has important ecological and economic values. Exploring its genetic background and phylogenetic direction will be conducive to its further exploitation and use, and promote the process of vegetation restoration in the dry hot river valley region. A genome assembly of approximately 719.88 Mb was achieved at the contig level, featuring a contig N50 of 53.74 Mb. Of this, 98.58% of gene sequences were organized into 13 pseudochromosomes. The A. odoratissima genome contained 96.96% of conserved genes, including 1,538 intact single-copy genes and 42 intact duplicated genes. It had an angiosperm palaeotripling event and the last whole genome duplication event occurred approximately 62.9 million years ago. A. odoratissima shares 8,936 gene families with five other legume species, while 1,420 gene families are unique to A. odoratissima. Under drought stress, photosynthesis was significantly inhibited to reduce water consumption, osmoregulatory substances were significantly increased to alleviate osmotic stress, and flavonoid accumulation was induced to enhance antioxidant capacity through the up-regulation of AoANS (Aod07G019900) gene expression, thereby improving drought tolerance. High-quality reference genomes generated through molecular studies are advancing research into the molecular mechanisms of A. odoratissima.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-025-07523-5.

The assessment of elasmobranch biodiversity in Thailand benefits greatly from the application of DNA barcoding, which helps mitigate the challenge posed by a shortage of expert taxonomists. Fragments of COI and ND2 mitochondrial DNA were examined, and the strengths and weaknesses of these two markers were compared. In this study, DNA products from 153 elasmobranch samples were amplifiable and revealed a total of 28 shark species and 32 batoid species. Many species could be confidently identified as their morphological characteristics aligned with DNA barcodes. However, several exceptions were recognized. The absence of reference sequences for rare species presented a challenge for species verification, and the misidentification of reference sequences, as well as changes in species names due to taxonomic revisions, added complexity when comparing DNA barcoding sequences. Conflicts between morphology and genetics were also observed. While intraspecific genetic variation based on both DNA barcodes generally indicated 0–2% variation, this metric could not always be used for species delimitation. This was particularly true for species displaying low genetic variation among closely related species and species where cryptic diversity remained hidden and yet to be uncovered. In such cases, the morphological characteristics of the samples served as the primary means of species identification. Despite these challenges, DNA barcoding remains an invaluable tool for biodiversity assessment, especially in light of the shortage of skilled experts, and for identification of products made from vulnerable species. However, it is essential to exercise caution and be aware of these complexities in its application.

Structural Variants (SVs) are defined as genomic variants affecting more than 500 base pairs. They include deletions, insertions, inversions, translocations, tandem repeats, and copy number variations (CNVs). These SVs contribute significantly to genetic complexity and are involved in human evolution, genetic disorders, and cancer. Over 50% of the SVs cannot be detected due to limitations in methods and technologies. The short-read sequencing technologies are limited in detecting single-nucleotide variants and fail to resolve complex, repetitive and intronic regions. The advent of long-read sequencing (LRS) technologies, such as Oxford Nanopore and PacBio platforms, has revolutionised SV detection. These platforms allow accurate characterisation of diverse variant types, from simple deletions to complex chromothripsis events, and support de novo assembly, haplotype phasing, and resolution of repetitive or structurally complex genomic regions. One major outcome is the completion of the telomere-to-telomere (T2T) human reference genome. This review summarises recent advances in LRS for SV detection, including sequencing platforms, bioinformatic tools, analysis, and validation strategies. The clinical applications, especially in rare disease diagnostics, are illustrated with two novel cases which were successfully resolved using both LRS approaches. Long-read sequencing can solve the limitations of short-read sequencing in SV detection, providing better insights into genome disorders. It enables the detection of repeat and difficult-to-resolve regions of the genome and facilitates clinical diagnoses to base-level breakpoint detection. Despite challenges such as high cost, data interpretation, and clinical linking, continued advancements are elevating LRS as an invaluable tool in precision genomic medicine.

Approximately half of high-grade serous ovarian carcinomas (HGSOCs) demonstrate homologous recombination deficiency (HRD) with characteristic genomic rearrangements. However, the impact of HRD on centromeres and transposable elements remains largely unexplored in HGSOC since conventional short-read sequencing is unable to interrogate these repetitive regions. We employed Oxford Nanopore long-read sequencing (LRS) to investigate genomic and epigenetic alterations in these regions. Pre-treatment archival cryopreserved tumor and matched blood samples were obtained for six patients with HGSOC. High-molecular-weight DNA was sequenced using Oxford Nanopore R10.4 flow cells and aligned to both the GRCh38 and the telomere-to-telomere T2T-CHM13 reference genome. Pathogenic gene mutations, allele-specific copy number variations, structural variants, and CpG methylation were analyzed. All six tumors had pathogenic TP53 mutations. Two carried germline BRCA1 mutations, while three showed CCNE1 amplifications. HRD scores and mutational signatures associated with HRD were elevated in the BRCA1-mutated tumors. Centromeric regions were significantly hypomethylated in tumors and their methylation profiles distinctly separated HRD tumors from non-HRD tumors. LINE1 and ERV transposable elements showed marked hypomethylation in tumors without germline BRCA1 mutations. Chromosome arm-specific telomere lengths were significantly shortened in tumors. Allele-specific hypermethylation in the TERT hypermethylated oncological region was detected in three tumors. LRS uncovered HRD-related genomic and epigenomic alterations in previously inaccessible repetitive regions of HGSOC, including centromeric and transposable element hypomethylation. These findings highlight the potential of such abnormalities as novel biomarkers for HGSOC and warrant further application of the methods to larger cohorts in future studies.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-21907-5.