Search Results (43)

Chinese pine, Pinus tabuliformis, is one of the most important garden plants in northern China, and the planting of this species is of great significance for the improvement of the ecological environment. In this study, different fungi were isolated and purified from diseased Pinus tabuliformis samples collected in Xi’an city, Shaanxi Province. Of these fungal isolates, only one (isolate AP-3) was pathogenic to the healthy host plant. The pathogenic isolate was identified as Fusarium acuminatum by morphological characteristics and ITS and TEF-1α sequence analyses. The optimal growth conditions for this isolate were further analyzed as follows: Optimal temperature of 25 °C, pH of 11, soluble starch and sodium nitrate as the most preferred carbon and nitrogen sources, respectively. By combining Oxford Nanopore Technologies (ONT) long-read sequencing with Illumina short-read sequencing technologies, we obtained a 41.50 Mb genome assembly for AP-3, with 47.97% GC content and 3.04% repeats. This consisted of 14 contigs with an N50 of 4.64 Mb and a maximum length of 6.45 Mb. The BUSCO completeness of the genome assembly was 98.94% at the fungal level and 97.83% at the Ascomycota level. The genome assembly contained 13,408 protein-coding genes, including 421 carbohydrate-active enzymes (CAZys), 120 cytochrome P450 enzymes (CYPs), 3185 pathogen-host interaction (PHI) genes, and 694 candidate secreted proteins. To our knowledge, this is the first report of F. acuminatum causing needle blight of P. tabuliformis. This study not only uncovered the pathogen responsible for needle blight of P. tabuliformis, but also provided a systematic analysis of its biological characteristics. These findings provide an important theoretical basis for disease control in P. tabuliformis and pave the way for further research into the fungal pathogenicity mechanisms and management strategies. Full article

Background: Structural variants (SVs) play a significant role in gene function and are implicated in numerous human diseases. With advances in sequencing technologies, identifying SVs through whole-genome sequencing (WGS) has become a key area of research. However, variability in SV detection persists due to the wide range of available tools and the absence of standardized methodologies. Methods: We assessed the accuracy of SV detection across various short-read (srWGS) and long-read (lrWGS) sequencing technologies—including Illumina short reads, PacBio long reads, and Oxford Nanopore Technologies (ONT) long reads—using deletion calls from the HG002 benchmark dataset. We examined how variables such as variant calling algorithms, reference genome choice, alignment strategies, and sequencing coverage influence SV detection performance. Results: DRAGEN v4.2 delivered the highest accuracy among ten srWGS callers tested. Notably, leveraging a graph-based multigenome reference improved SV calling in complex genomic regions. Moreover, we proved that combining minimap2 with Manta achieved performance comparable to DRAGEN for srWGS. For PacBio lrWGS data, Sniffles2 outperformed the other two tested tools. For ONT lrWGS, alignment with minimap2—among four aligners tested—consistently led to the best results. At up to 10× coverage, Duet achieved the highest accuracy, while at higher coverages, Dysgu yielded the best results. Conclusions: These results show for the first time that alignment software choice significantly impacts SV calling from srWGS, with results comparable to commercial solutions. For lrWGS, the performance depends on the technology and coverage. Full article

Wastewater-based epidemiology (WBE) offers valuable insight into viral circulation at the community level. In this study, we combined digital PCR (dPCR) with molecular typing to investigate the prevalence and diversity of human adenoviruses (HAdVs) in untreated wastewater samples collected throughout Italy. HAdV genomes were detected in over 93% of the 168 samples analyzed, with concentrations up to 4.5 × 10⁶ genome copies per liter. For genotypic characterization, we used nested PCR followed by Sanger and Oxford Nanopore Technologies (ONTs) long-read sequencing. While Sanger sequencing identified three dominant genotypes (HAdV-A12, HAdV-B3, and HAdV-F41), ONT sequencing provided enhanced resolution, confirming all previously identified types and revealing seven additional genotypes: HAdV-B21, HAdV-C5, HAdV-D45, HAdV-D46, HAdV-D49, HAdV-D83, and HAdV-F40. This comprehensive approach highlights the added value of ONT long-read sequencing in uncovering the genetic complexity of adenoviruses in wastewater, particularly in detecting rare or low abundance types that conventional methods may miss. Our findings highlight the value of integrating quantitative and high-resolution molecular tools in WBE to improve surveillance and better understand the epidemiology of viral pathogens circulating in the human population. Full article

Oxford Nanopore Technologies (ONT) long-read sequencing (LRS) has emerged as a promising genomic analysis tool, yet comprehensive benchmarks with established platforms across diverse datasets remain limited. This study aimed to benchmark LRS performance against Illumina short-read sequencing (SRS) and microarrays for variant detection across different genomic contexts and to evaluate the impact of experimental factors. We sequenced 14 human genomes using the three platforms and evaluated single nucleotide variants (SNVs), insertions/deletions (indels), and structural variants (SVs) detection, stratifying by high-complexity, low-complexity, and dark genome regions while assessing effects of multiplexing, depth, and read length. LRS SNV accuracy was slightly lower than that of SRS in high-complexity regions (F-measure: 0.954 vs. 0.967) but showed comparable sensitivity in low-complexity regions. LRS showed robust performance for small (1–5 bp) indels in high-complexity regions (F-measure: 0.869), but SRS agreement decreased significantly in low-complexity regions and for larger indel sizes. Within dark regions, LRS identified more indels than SRS, but showed lower base-level accuracy. LRS identified 2.86 times more SVs than SRS, excelling at detecting large variants (>6 kb), with SV detection improving with sequencing depth. Sequencing depth strongly influenced variant calling performance, whereas multiplexing effects were minimal. Our findings provide valuable insights for optimising LRS applications in genomic research and diagnostics. Full article

While whole-genome sequencing (WGS) using short-read technology has become a standard diagnostic test, this technology has limitations in analyzing certain genomic regions, particularly short tandem repeats (STRs). These repetitive sequences are associated with over 50 diseases, primarily affecting neurological function, including Huntington disease, frontotemporal dementia, and Friedreich’s ataxia. We analyzed 2689 cases with movement disorders and dementia-related phenotypes processed at Variantyx in 2023–2024 using a two-tiered approach, with an initial short-read WGS followed by ONT long-read sequencing (when necessary) for variant characterization. Of the 2038 cases (75.8%) with clinically relevant genetic variants, 327 (16.0%) required additional long-read analysis. STR variants were reported in 338 cases (16.6% of positive cases), with approximately half requiring long-read sequencing for definitive classification. The combined approach enabled the precise determination of repeat length, composition, somatic mosaicism, and methylation status. Notable advantages included the detection of complex repeat structures in several genes such as RFC1, FGF14, and FXN, where long-read sequencing allowed to determine somatic repeat unit variations and accurate allele phasing. Further studies are needed to establish technology-specific guidelines for the standardized interpretation of long-read sequencing data for the clinical diagnostics of repeat expansion disorders. Full article

Background/Objectives: Massively parallel sequencing technologies have advanced chronic lymphocytic leukemia (CLL) diagnostics and precision oncology. Illumina platforms, while offering robust performance, require substantial infrastructure investment and a large number of samples for cost-efficiency. Conversely, third-generation long-read nanopore sequencing from Oxford Nanopore Technologies (ONT) can significantly reduce sequencing costs, making it a valuable tool in resource-limited settings. However, nanopore sequencing faces challenges with lower accuracy and throughput than Illumina platforms, necessitating additional computational strategies. In this paper, we demonstrate that integrating publicly available short-read data with in-house generated ONT data, along with the application of machine learning approaches, enables the characterization of the CLL transcriptome landscape, the identification of clinically relevant molecular subtypes, and the assignment of these subtypes to nanopore-sequenced samples. Methods: Public Illumina RNA sequencing data for 608 CLL samples were obtained from the CLL-Map Portal. CLL transcriptome analysis, gene module identification, and transcriptomic subtype classification were performed using the oposSOM R package for high-dimensional data visualization with self-organizing maps. Eight CLL patients were recruited from the Hematology Center After Prof. R. Yeolyan (Yerevan, Armenia). Sequencing libraries were prepared from blood total RNA using the PCR-cDNA sequencing-barcoding kit (SQK-PCB109) following the manufacturer’s protocol and sequenced on an R9.4.1 flow cell for 24–48 h. Raw reads were converted to TPM values. These data were projected into the SOMs space using the supervised SOMs portrayal (supSOM) approach to predict the SOMs portrait of new samples using support vector machine regression. Results: The CLL transcriptomic landscape reveals disruptions in gene modules (spots) associated with T cell cytotoxicity, B and T cell activation, inflammation, cell cycle, DNA repair, proliferation, and splicing. A specific gene module contained genes associated with poor prognosis in CLL. Accordingly, CLL samples were classified into T-cell cytotoxic, immune, proliferative, splicing, and three mixed types: proliferative–immune, proliferative–splicing, and proliferative–immune–splicing. These transcriptomic subtypes were associated with survival orthogonal to gender and mutation status. Using supervised machine learning approaches, transcriptomic subtypes were assigned to patient samples sequenced with nanopore sequencing. Conclusions: This study demonstrates that the CLL transcriptome landscape can be parsed into functional modules, revealing distinct molecular subtypes based on proliferative and immune activity, with important implications for prognosis and treatment that are orthogonal to other molecular classifications. Additionally, the integration of nanopore sequencing with public datasets and machine learning offers a cost-effective approach to molecular subtyping and prognostic prediction, facilitating more accessible and personalized CLL care. Full article

This study presents a comprehensive analysis of mitochondrial DNA (mtDNA) variations in dogs diagnosed with primary and recurrent tumours, employing Oxford Nanopore Technologies (ONT) for sequencing. Our investigation focused on mtDNA extracted from blood and tumour tissues of three dogs, aiming to pinpoint polymorphisms, mutations, and heteroplasmy levels that could influence mitochondrial function in cancer pathogenesis. Notably, we observed the presence of mutations in the D-loop region, especially in the VNTR region, which may be crucial for mitochondrial replication, transcription, and genome stability, suggesting its potential role in cancer progression. The study is pioneering in its use of long-read sequencing to explore the mutational landscape of mtDNA in canine tumours, revealing that while the overall mutational load did not differ between primary and recurrent tumours, specific changes in m.16168A/G, m.16188G/A, and m.16298A/G are linked with tumour tissues. Interestingly, the heteroplasmy outside the D-loop region was not specific to tumour tissues and did not provoke any malignant damage in protein-coding sequences, which in turn may be a tolerant effect of the reactive oxygen species (ROS) cellular stress mechanism. Full article

Flax is an important crop grown for seed and fiber. Flax chromosome number is 2n = 30, and its genome size is about 450–480 Mb. To date, the genomes of several flax varieties have been sequenced and assembled. However, the obtained assemblies are still far from the telomere-to-telomere (T2T) level. We sequenced the genome of flax variety K-3018 on the Oxford Nanopore Technologies (ONT) platform and obtained 57.7 Gb of R10 simplex reads with an N50 = 18.4 kb (~120× genome coverage). ONT reads longer than 50 kb were kept as ultra-long ones (~10× genome coverage), and the rest of the ONT reads were corrected using the HERRO R10 model (quality > Q10, length > 10 kb, ~60× genome coverage remained). The genome was assembled using Hifiasm and Verkko. The Hifiasm-generated assembly was 489.1 Mb in length with 54 contigs and an N50 = 28.1 Mb. Verkko produced a very similar but more fragmented genome: 489.1 Mb, 134 contigs, N50 = 17.4 Mb. In the assembly by Hifiasm, eight chromosomes consisted of a single contig with telomeric repeats at both ends. In addition, five chromosomes comprised two contigs and two chromosomes comprised three contigs. These chromosomes also had telomeric repeats at their ends. The Hifiasm-generated assembly of variety K-3018 had similar contiguity but was likely more complete and accurate than the main fifteen-chromosome assembly of variety YY5 (produced from PacBio data and scaffolded with Hi-C data), the most contiguous flax genome assembly at the time of this writing. We suggest that sufficient genome coverage with long ONT R10 simplex reads is a viable alternative to PacBio plus Hi-C data for a high-precision T2T genome assembly of flax, opening new perspectives for whole-genome studies of flax. Full article

Background. In the context of increasing antimicrobial resistance (AMR), whole-genome sequencing (WGS) of bacteria is considered a highly accurate and comprehensive surveillance method for detecting and tracking the spread of resistant pathogens. Two primary sequencing technologies exist: short-read sequencing (50–300 base pairs) and long-read sequencing (thousands of base pairs). The former, based on Illumina sequencing platforms (ISPs), provides extensive coverage and high accuracy for detecting single nucleotide polymorphisms (SNPs) and small insertions/deletions, but is limited by its read length. The latter, based on platforms such as Oxford Nanopore Technologies (ONT), enables the assembly of genomes, particularly those with repetitive regions and structural variants, although its accuracy has historically been lower. Results. We performed a head-to-head comparison of these techniques to sequence the K. pneumoniae VS17 isolate, focusing on bla_NDM resistance gene alleles in the context of a surveillance program. Discrepancies between the ISP (bla_NDM-4 allele identified) and ONT (bla_NDM-1 and bla_NDM-5 alleles identified) were observed. Conjugation assays and Sanger sequencing, used as the gold standard, confirmed the validity of ONT results. This study demonstrates the importance of long-read or hybrid assemblies for accurate carbapenemase resistance gene identification and highlights the limitations of short reads in the context of gene duplications or multiple alleles. Conclusions. In this proof-of-concept study, we conclude that recent long-read sequencing technology may outperform standard short-read sequencing for the accurate identification of carbapenemase alleles. Such information is crucial given the rising prevalence of strains producing multiple carbapenemases, especially as WGS is increasingly used for epidemiological surveillance and infection control. Full article

Lichens have developed numerous adaptations to optimize their survival in various environmental conditions, largely by producing secondary compounds by the fungal partner. They often have antibiotic properties and are involved in protection against intensive UV radiation, pathogens, and herbivores. To contribute to the knowledge of the arsenal of secondary compounds in a crustose lichen species, we sequenced and assembled the genome of Toniniopsis dissimilis, an indicator of old-growth forests, using Oxford Nanopore Technologies (ONT, Oxford, UK) long reads. Our analyses focused on biosynthetic gene clusters (BGCs) and specifically on Type I Polyketide (T1PKS) genes involved in the biosynthesis of polyketides. We used the comparative genomic approach to compare the genome of T. dissimilis with six other members of the family Ramalinaceae and twenty additional lichen genomes from the database. With only six T1PKS genes, a comparatively low number of biosynthetic genes are present in the T. dissimilis genome; from those, two-thirds are putatively involved in melanin biosynthesis. The comparative analyses showed at least three potential pathways of melanin biosynthesis in T. dissimilis, namely via the formation of 1,3,6,8-tetrahydroxynaphthalene, naphthopyrone, or YWA1 putative precursors, which highlights its importance in T. dissimilis. In addition, we report the occurrence of genes encoding ribosomally synthesized and posttranslationally modified peptides (RiPPs) in lichens, with their highest number in T. dissimilis compared to other Ramalinaceae genomes. So far, no function has been assigned to RiPP-like proteins in lichens, which leaves potential for future research on this topic. Full article

The identification of structural variants (SVs) in genomic data represents an ongoing challenge because of difficulties in reliable SV calling leading to reduced sensitivity and specificity. We prepared high-quality DNA from 9 parent–child trios, who had previously undergone short-read whole-genome sequencing (Illumina platform) as part of the Genomics England 100,000 Genomes Project. We reanalysed the genomes using both Bionano optical genome mapping (OGM; 8 probands and one trio) and Nanopore long-read sequencing (Oxford Nanopore Technologies [ONT] platform; all samples). To establish a “truth” dataset, we asked whether rare proband SV calls (n = 234) made by the Bionano Access (version 1.6.1)/Solve software (version 3.6.1_11162020) could be verified by individual visualisation using the Integrative Genomics Viewer with either or both of the Illumina and ONT raw sequence. Of these, 222 calls were verified, indicating that Bionano OGM calls have high precision (positive predictive value 95%). We then asked what proportion of the 222 true Bionano SVs had been identified by SV callers in the other two datasets. In the Illumina dataset, sensitivity varied according to variant type, being high for deletions (115/134; 86%) but poor for insertions (13/58; 22%). In the ONT dataset, sensitivity was generally poor using the original Sniffles variant caller (48% overall) but improved substantially with use of Sniffles2 (36/40; 90% and 17/23; 74% for deletions and insertions, respectively). In summary, we show that the precision of OGM is very high. In addition, when applying the Sniffles2 caller, the sensitivity of SV calling using ONT long-read sequence data outperforms Illumina sequencing for most SV types. Full article

The marine nematode Litoditis marina is widely distributed in intertidal zones around the globe, yet the mechanisms underlying its broad adaptation to salinity remain elusive. In this study, we applied ONT long-read sequencing technology to unravel the transcriptome responses to different salinity conditions in L. marina. Through ONT sequencing under 3‰, 30‰ and 60‰ salinity environments, we obtained 131.78 G clean data and 26,647 non-redundant long-read transcripts, including 6464 novel transcripts. The DEGs obtained from the current ONT lrRNA-seq were highly correlated with those identified in our previously reported Illumina short-read RNA sequencing data. When we compared the 30‰ to the 3‰ salinity condition, we found that GO terms such as oxidoreductase activity, cation transmembrane transport and ion transmembrane transport were shared between the ONT lrRNA-seq and Illumina data. Similarly, GO terms including extracellular space, structural constituents of cuticle, substrate-specific channel activity, ion transport and substrate-specific transmembrane transporter activity were shared between the ONT and Illumina data under 60‰ compared to 30‰ salinity. In addition, we found that 79 genes significantly increased, while 119 genes significantly decreased, as the salinity increased. Furthermore, through the GO enrichment analysis of 214 genes containing DAS, in 30‰ compared to 3‰ salinity, we found that GO terms such as cellular component assembly and coenzyme biosynthetic process were enriched. Additionally, we observed that GO terms such as cellular component assembly and coenzyme biosynthetic process were also enriched in 60‰ compared to 30‰ salinity. Moreover, we found that 86, 125, and 81 genes that contained DAS were also DEGs, in comparisons between 30‰ and 3‰, 60‰ and 30‰, and 60‰ and 3‰ salinity, respectively. In addition, we demonstrated the landscape of alternative polyadenylation in marine nematode under different salinity conditions This report provides several novel insights for the further study of the mechanisms by which euryhalinity formed and evolved, and it might also contribute to the investigation of salinity dynamics induced by global climate change. Full article

Third-generation sequencing technology has found widespread application in the genomic, transcriptomic, and epigenetic research of both human and livestock genetics. This technology offers significant advantages in the sequencing of complex genomic regions, the identification of intricate structural variations, and the production of high-quality genomes. Its attributes, including long sequencing reads, obviation of PCR amplification, and direct determination of DNA/RNA, contribute to its efficacy. This review presents a comprehensive overview of third-generation sequencing technologies, exemplified by single-molecule real-time sequencing (SMRT) and Oxford Nanopore Technology (ONT). Emphasizing the research advancements in livestock genomics, the review delves into genome assembly, structural variation detection, transcriptome sequencing, and epigenetic investigations enabled by third-generation sequencing. A comprehensive analysis is conducted on the application and potential challenges of third-generation sequencing technology for genome detection in livestock. Beyond providing valuable insights into genome structure analysis and the identification of rare genes in livestock, the review ventures into an exploration of the genetic mechanisms underpinning exemplary traits. This review not only contributes to our understanding of the genomic landscape in livestock but also provides fresh perspectives for the advancement of research in this domain. Full article

Antimalarial drug resistance has become a real public health problem despite WHO measures. New sequencing technologies make it possible to investigate genomic variations associated with resistant phenotypes at the genome-wide scale. Based on the use of hemisynthetic nanopores, the PromethION technology from Oxford Nanopore Technologies can produce long-read sequences, in contrast to previous short-read technologies used as the gold standard to sequence Plasmodium. Two clones of P. falciparum (Pf3D7 and PfW2) were sequenced in long-read using the PromethION sequencer from Oxford Nanopore Technologies without genomic amplification. This made it possible to create a processing analysis pipeline for human Plasmodium with ONT Fastq only. De novo assembly revealed N50 lengths of 18,488 kb and 17,502 kb for the Pf3D7 and PfW2, respectively. The genome size was estimated at 23,235,407 base pairs for the Pf3D7 clone and 21,712,038 base pairs for the PfW2 clone. The average genome coverage depth was estimated at 787X and 653X for the Pf3D7 and PfW2 clones, respectively. This study proposes an assembly processing pipeline for the human Plasmodium genome using software adapted to large ONT data and the high AT percentage of Plasmodium. This search provides all the parameters which were optimized for use with the software selected in the pipeline. Full article

The sea star Patiria pectinifera (Asteroidea; Asterinidae; homotypic synonym: Asterina pectinifera; Muller & Troschel, 1842) is widely distributed in the coastal regions of the Seas of East Asia and the northern Pacific Ocean. Here, a de novo genome sequence of P. pectinifera as a reference for fundamental and applied research was constructed by employing a combination of long-read Oxford Nanopore Technology (ONT) PromethION, short-read Illumina platforms, and 10 × Genomics. The draft genome of P. pectinifera, containing 13,848,344 and 156,878,348 contigs from ONT and Illumina platforms, respectively, was obtained. Assembly with CANU resulted in 2262 contigs with an N50 length of 367 kb. Finally, ARCS + LINKS assembly combined these contigs into 328 scaffolds, totaling 499 Mb with an N50 length of 2 Mbp. The estimated genome size by GenomeScope analysis was 461 Mb. BUSCO analysis indicated that 930 (97.5%) of the expected genes were found in the assembly, with 889 (93.2%) being single-copy and 41 (4.3%) duplicated after searching against the metazoan database. Annotation, utilizing sequences obtained from Illumina RNA-Seq and Pacific Biosciences Iso-Seq, led to the identification of 22,367 protein-coding genes. When examining the orthologous relationship of P. pectinifera against the scaffolds of the common sea star Patiria miniata, high contiguity was observed. Annotation of repeat elements highlighted an enrichment of 1,121,079 transposable elements, constituting 47% of the genome, suggesting their potential role in shaping the genome structure of P. pectinifera. This de novo genome assembly is expected to be a valuable resource for future studies, providing insight into the developmental, environmental, and ecological aspects of P. pectinifera biology. Full article