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Keywords = single-molecule long-read sequencing

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19 pages, 1240 KiB  
Review
Evaluation of Illumina and Oxford Nanopore Sequencing for the Study of DNA Methylation in Alzheimer’s Disease and Frontotemporal Dementia
by Lorenzo Pagano, Davide Lagrotteria, Alessandro Facconi, Claudia Saraceno, Antonio Longobardi, Sonia Bellini, Assunta Ingannato, Silvia Bagnoli, Tommaso Ducci, Alessandra Mingrino, Valentina Laganà, Ersilia Paparazzo, Barbara Borroni, Raffaele Maletta, Benedetta Nacmias, Alberto Montesanto and Roberta Ghidoni
Int. J. Mol. Sci. 2025, 26(9), 4198; https://doi.org/10.3390/ijms26094198 - 28 Apr 2025
Cited by 1 | Viewed by 1383
Abstract
DNA methylation is a critical epigenetic mechanism involved in numerous physiological processes. Alterations in DNA methylation patterns are associated with various brain disorders, including dementias such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Investigating these alterations is essential for understanding the pathogenesis [...] Read more.
DNA methylation is a critical epigenetic mechanism involved in numerous physiological processes. Alterations in DNA methylation patterns are associated with various brain disorders, including dementias such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD). Investigating these alterations is essential for understanding the pathogenesis and progression of these disorders. Among the various methods for detecting DNA methylation, DNA sequencing is one of the most widely employed. Specifically, two main sequencing approaches are commonly used for DNA methylation analysis: bisulfite sequencing and single-molecule long-read sequencing. In this review, we compared the performances of CpG methylation detection obtained using two popular sequencing platforms, Illumina for bisulfite sequencing and Oxford Nanopore (ON) for long-read sequencing. Our comparison considers several factors, including accuracy, efficiency, genomic regions, costs, wet-lab protocols, and bioinformatics pipelines. We provide insights into the strengths and limitations of both methods with a particular focus on their application in research on AD and FTD. Full article
(This article belongs to the Special Issue New Advances in Epigenetics and Epigenomics)
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13 pages, 1818 KiB  
Hypothesis
The Origin of Life in the Early Continental Crust: A Comprehensive Model
by Ulrich Schreiber
Life 2025, 15(3), 433; https://doi.org/10.3390/life15030433 - 10 Mar 2025
Viewed by 1386
Abstract
Continental rift zones on the early Earth provided essential conditions for the emergence of the first cells. These conditions included an abundant supply of raw materials, cyclic fluctuations in pressure and temperature over millions of years, and transitions of gases between supercritical and [...] Read more.
Continental rift zones on the early Earth provided essential conditions for the emergence of the first cells. These conditions included an abundant supply of raw materials, cyclic fluctuations in pressure and temperature over millions of years, and transitions of gases between supercritical and subcritical phases. While evidence supports vesicle formation and the chemical evolution of peptides, the mechanism by which information was stored remains unresolved. This study proposes a model illustrating how interactions among organic molecules may have enabled the encoding of amino acid sequences in RNA. The model highlights the interplay between three key molecular components: a proto-tRNA, the vesicle membrane, and short peptides. The vesicle membrane acted as a reservoir for hydrophobic amino acids and facilitated their attachment to proto-tRNA. As a single strand, proto-tRNA also served as proto-mRNA, enabling it to be read by charged tRNAs. By replicating this information and arranging RNA strands, the first functional peptides such as pore-forming proteins may have formed, thus improving the long-term stability of the vesicles. This model further outlines how these vesicles may have evolved into the earliest cells, with enzymes and larger RNA molecules giving rise to tRNA and ribosomal structures. Shearing forces may have facilitated the first cellular divisions, representing a pre-LUCA stage. Full article
(This article belongs to the Special Issue 2nd Edition—Featured Papers on the Origins of Life)
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13 pages, 1850 KiB  
Article
Bioinformatics Tools for NGS-Based Identification of Single Nucleotide Variants and Large-Scale Rearrangements in Mitochondrial DNA
by Marco Barresi, Giulia Dal Santo, Rossella Izzo, Andrea Zauli, Eleonora Lamantea, Leonardo Caporali, Daniele Ghezzi and Andrea Legati
BioTech 2025, 14(1), 9; https://doi.org/10.3390/biotech14010009 - 12 Feb 2025
Viewed by 1294
Abstract
The unique features of mitochondrial DNA (mtDNA), including its circular and multicopy nature, the possible coexistence of wild-type and mutant molecules (i.e., heteroplasmy) and the presence of nuclear mitochondrial DNA segments (NUMTs), make the diagnosis of mtDNA diseases particularly challenging. The extensive deployment [...] Read more.
The unique features of mitochondrial DNA (mtDNA), including its circular and multicopy nature, the possible coexistence of wild-type and mutant molecules (i.e., heteroplasmy) and the presence of nuclear mitochondrial DNA segments (NUMTs), make the diagnosis of mtDNA diseases particularly challenging. The extensive deployment of next-generation sequencing (NGS) technologies has significantly advanced the diagnosis of mtDNA-related diseases. However, the vast amounts and diverse types of sequencing data complicate the interpretation of these variants. From sequence alignment to variant calling, NGS-based mtDNA sequencing requires specialized bioinformatics tools, adapted for the mitochondrial genome. This study presents the use of new bioinformatics approaches, optimized for short- and long-read sequencing data, to enhance the accuracy of mtDNA analysis in diagnostics. Two recent and emerging free bioinformatics tools, Mitopore and MitoSAlt, were evaluated on patients previously diagnosed with single nucleotide variants or large-scale deletions. Analyses were performed in Linux-based environments and web servers implemented in Python, Perl, Java, and R. The results indicated that each tool demonstrated high sensitivity and specific accuracy in identifying and quantifying various types of pathogenic variants. The study suggests that the integrated and parallel use of these tools offers a significant advantage over traditional methods in interpreting mtDNA genetic variants, reducing the computational demands, and provides an accurate diagnostic solution. Full article
(This article belongs to the Section Computational Biology)
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18 pages, 2372 KiB  
Article
Genome De Novo (WGS) Sequence Resource of the Lasiodiplodia theobromae Bot-2018-LT45 Isolate Causing Dieback in Apple
by Adrián V. Valdez-Tenezaca, Sergio A. Hernández Covarrubias, Alexis G. Murillo Carrasco, Matías I. Guerra Peñalosa, Jean F. Castro Figueroa, M. Ernesto Delgado Fernández, José A. Corona-Gómez and Gonzalo A. Díaz Ulloa
Int. J. Plant Biol. 2025, 16(1), 10; https://doi.org/10.3390/ijpb16010010 - 9 Jan 2025
Viewed by 1334
Abstract
Lasiodiplodia theobromae is a pathogenic fungus associated with tropical perennial fruit plants worldwide. In apple trees, L. theobromae causes dieback and canker, a disease that affects the architecture of the wood producing the progressive death of branches and stems, from the tips to [...] Read more.
Lasiodiplodia theobromae is a pathogenic fungus associated with tropical perennial fruit plants worldwide. In apple trees, L. theobromae causes dieback and canker, a disease that affects the architecture of the wood producing the progressive death of branches and stems, from the tips to the base, invading the vascular tissue, manifesting necrotic lesions in the bark, impeding the flow of nutrients and water. The present work reports the whole genome de novo sequencing (WGS) of L. theobromae strain Bot-2018-LT45 isolated from apple trees with dieback symptoms. Genomic DNA of L. theobromae was sequenced using Illumina paired-end short-read technology (NovaSeq6000) and PacBio SMRTbellTM (Single Molecule, Real-Time) long-read technology. The genome size was 44.17 Mb. Then, assembly and annotation revealed a total of 12,948 genes of which 11,634 encoded proteins. The genome was assembled into 34 contigs with an N50 (Mb) value of 3.23. This study is the first report of the L. theobromae genome de novo obtained from apple trees with dieback and canker symptoms in the Maule Region, Chile. This genetic information may set the basis for future study of the mechanisms of L. theobromae and establish the possibility of specific molecular improvements for the control of dieback and canker. Full article
(This article belongs to the Section Plant Biochemistry and Genetics)
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18 pages, 2344 KiB  
Review
The Research Progress of Single-Molecule Sequencing and Its Significance in Nucleic Acid Metrology
by Yajun Wang, Jingjing Liu, Zhendong Wang, Mei Zhang and Yongzhuo Zhang
Biosensors 2025, 15(1), 4; https://doi.org/10.3390/bios15010004 - 25 Dec 2024
Viewed by 1935
Abstract
Single-molecule sequencing technology, a novel method for gene sequencing, utilizes nano-sized materials to detect electrical and fluorescent signals. Compared to traditional Sanger sequencing and next-generation sequencing technologies, it offers significant advantages, including ultra-long read lengths, rapid sequencing, and the absence of amplification steps, [...] Read more.
Single-molecule sequencing technology, a novel method for gene sequencing, utilizes nano-sized materials to detect electrical and fluorescent signals. Compared to traditional Sanger sequencing and next-generation sequencing technologies, it offers significant advantages, including ultra-long read lengths, rapid sequencing, and the absence of amplification steps, making it widely applicable across various fields. By examining the development and components of single-molecule sequencing technology, it becomes clear that its unique characteristics provide new opportunities for advancing metrological traceability. Notably, its direct detection capabilities offer a novel approach to nucleic acid metrology. This paper provides a detailed overview of library construction, signal generation and detection, and data analysis methods in single-molecule sequencing and discusses its implications for nucleic acid metrology. Full article
(This article belongs to the Special Issue Micro-nano Optic-Based Biosensing Technology and Strategy)
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16 pages, 6388 KiB  
Article
Comparative Analysis of Alternative Splicing in Moso Bamboo and Its Dwarf Mutant, Phyllostachys edulisTubaeformis
by Zhenhua Qiu, Yuanyuan Sun, Yanhui Su, Long Cheng, Dong Liu, Shuyan Lin and Long Li
Forests 2024, 15(7), 1233; https://doi.org/10.3390/f15071233 - 15 Jul 2024
Viewed by 1271
Abstract
Internode length is a crucial phenotypic trait of bamboo, significantly impacting its processing and utilization. Phyllostachys edulisTubaeformis’ (Shengyin Bamboo), a variety of Moso bamboo, exhibits drastically shortened internodes, making it a valuable ornamental bamboo species. We used PacBio single-molecule long-read [...] Read more.
Internode length is a crucial phenotypic trait of bamboo, significantly impacting its processing and utilization. Phyllostachys edulisTubaeformis’ (Shengyin Bamboo), a variety of Moso bamboo, exhibits drastically shortened internodes, making it a valuable ornamental bamboo species. We used PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and its dwarf mutant, Shengyin bamboo, and compared the differences between the two. Our sequencing data unveiled 139,539 AS events, with retained introns as the most prevalent events. A large number of genes were differentially alternatively spliced (DAS) between Moso bamboo and Shengyin bamboo, and genes related to RNA splicing were most significantly enriched. The high expression of SR isoforms in the 24th internode of Moso bamboo is likely the main factor leading to its greater number of alternative splicing events. Alternative splicing affects the functional domains of partial GRF, E2F, and NAM isoforms, leading to the loss of domains in some isoforms and enabling some isoforms to acquire new functional domains, and this phenomenon is more common in Shengyin bamboo. AS modifies the functional domains of certain GRF isoforms, frequently resulting in domain losses or endowing isoforms with novel domains, and this phenomenon is more common in Shengyin bamboo. We used PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and its dwarf mutant, Shengyin Bamboo and compared the differences between the two. Full article
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12 pages, 4483 KiB  
Article
Transcriptional Regulation Analysis Provides Insight into the Function of GSK3β Gene in Diannan Small-Ear Pig Spermatogenesis
by Xia Zhang, Guiying Zhao, Fuhua Yang, Changyao Li, Wan Lin, Hongmei Dai, Lan Zhai, Xuemin Xi, Qingting Yuan and Jinlong Huo
Genes 2024, 15(6), 655; https://doi.org/10.3390/genes15060655 - 22 May 2024
Viewed by 1813
Abstract
Glycogen synthase kinase-3β (GSK3β) not only plays a crucial role in regulating sperm maturation but also is pivotal in orchestrating the acrosome reaction. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine GSK3β expression patterns in adult Diannan small-ear pig (DSE) [...] Read more.
Glycogen synthase kinase-3β (GSK3β) not only plays a crucial role in regulating sperm maturation but also is pivotal in orchestrating the acrosome reaction. Here, we integrated single-molecule long-read and short-read sequencing to comprehensively examine GSK3β expression patterns in adult Diannan small-ear pig (DSE) testes. We identified the most important transcript ENSSSCT00000039364 of GSK3β, obtaining its full-length coding sequence (CDS) spanning 1263 bp. Gene structure analysis located GSK3β on pig chromosome 13 with 12 exons. Protein structure analysis reflected that GSK3β consisted of 420 amino acids containing PKc-like conserved domains. Phylogenetic analysis underscored the evolutionary conservation and homology of GSK3β across different mammalian species. The evaluation of the protein interaction network, KEGG, and GO pathways implied that GSK3β interacted with 50 proteins, predominantly involved in the Wnt signaling pathway, papillomavirus infection, hippo signaling pathway, hepatocellular carcinoma, gastric cancer, colorectal cancer, breast cancer, endometrial cancer, basal cell carcinoma, and Alzheimer’s disease. Functional annotation identified that GSK3β was involved in thirteen GOs, including six molecular functions and seven biological processes. ceRNA network analysis suggested that DSE GSK3β was regulated by 11 miRNA targets. Furthermore, qPCR expression analysis across 15 tissues highlighted that GSK3β was highly expressed in the testis. Subcellular localization analysis indicated that the majority of the GSK3β protein was located in the cytoplasm of ST (swine testis) cells, with a small amount detected in the nucleus. Overall, our findings shed new light on GSK3β’s role in DSE reproduction, providing a foundation for further functional studies of GSK3β function. Full article
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19 pages, 8889 KiB  
Article
Divergence within the Taxon ‘Candidatus Phytoplasma asteris’ Confirmed by Comparative Genome Analysis of Carrot Strains
by Rafael Toth, Anna-Marie Ilic, Bruno Huettel, Bojan Duduk and Michael Kube
Microorganisms 2024, 12(5), 1016; https://doi.org/10.3390/microorganisms12051016 - 17 May 2024
Cited by 2 | Viewed by 2109
Abstract
Phytoplasmas are linked to diseases in hundreds of economically important crops, including carrots. In carrots, phytoplasmosis is associated with leaf chlorosis and necrosis, coupled with inhibited root system development, ultimately leading to significant economic losses. During a field study conducted in Baden-Württemberg (Germany), [...] Read more.
Phytoplasmas are linked to diseases in hundreds of economically important crops, including carrots. In carrots, phytoplasmosis is associated with leaf chlorosis and necrosis, coupled with inhibited root system development, ultimately leading to significant economic losses. During a field study conducted in Baden-Württemberg (Germany), two strains of the provisional taxon ‘Candidatus Phytoplasma asteris’ were identified within a carrot plot. For further analysis, strains M8 and M33 underwent shotgun sequencing, utilising single-molecule-real-time (SMRT) long-read sequencing and sequencing-by-synthesis (SBS) paired-end short-read sequencing techniques. Hybrid assemblies resulted in complete de novo assemblies of two genomes harboring circular chromosomes and two plasmids. Analyses, including average nucleotide identity and sequence comparisons of established marker genes, confirmed the phylogenetic divergence of ‘Ca. P. asteris’ and a different assignment of strains to the 16S rRNA subgroup I-A for M33 and I-B for M8. These groups exhibited unique features, encompassing virulence factors and genes, associated with the mobilome. In contrast, pan-genome analysis revealed a highly conserved gene set related to metabolism across these strains. This analysis of the Aster Yellows (AY) group reaffirms the perception of phytoplasmas as bacteria that have undergone extensive genome reduction during their co-evolution with the host and an increase of genome size by mobilome. Full article
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16 pages, 1405 KiB  
Review
When Livestock Genomes Meet Third-Generation Sequencing Technology: From Opportunities to Applications
by Xinyue Liu, Junyuan Zheng, Jialan Ding, Jiaxin Wu, Fuyuan Zuo and Gongwei Zhang
Genes 2024, 15(2), 245; https://doi.org/10.3390/genes15020245 - 15 Feb 2024
Cited by 2 | Viewed by 2845
Abstract
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 [...] Read more.
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
(This article belongs to the Special Issue Genetic Regulation of Animal Reproduction)
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14 pages, 2268 KiB  
Article
Viral Diversity in Benthic Abyssal Ecosystems: Ecological and Methodological Considerations
by Umberto Rosani, Cinzia Corinaldesi, Gabriella Luongo, Marco Sollitto, Simeone Dal Monego, Danilo Licastro, Lucia Bongiorni, Paola Venier, Alberto Pallavicini and Antonio Dell’Anno
Viruses 2023, 15(12), 2282; https://doi.org/10.3390/v15122282 - 21 Nov 2023
Viewed by 1450
Abstract
Viruses are the most abundant ‘biological entities’ in the world’s oceans. However, technical and methodological constraints limit our understanding of their diversity, particularly in benthic abyssal ecosystems (>4000 m depth). To verify advantages and limitations of analyzing virome DNA subjected either to random [...] Read more.
Viruses are the most abundant ‘biological entities’ in the world’s oceans. However, technical and methodological constraints limit our understanding of their diversity, particularly in benthic abyssal ecosystems (>4000 m depth). To verify advantages and limitations of analyzing virome DNA subjected either to random amplification or unamplified, we applied shotgun sequencing-by-synthesis to two sample pairs obtained from benthic abyssal sites located in the North-eastern Atlantic Ocean at ca. 4700 m depth. One amplified DNA sample was also subjected to single-molecule long-read sequencing for comparative purposes. Overall, we identified 24,828 viral Operational Taxonomic Units (vOTUs), belonging to 22 viral families. Viral reads were more abundant in the amplified DNA samples (38.5–49.9%) compared to the unamplified ones (4.4–5.8%), with the latter showing a greater viral diversity and 11–16% of dsDNA viruses almost undetectable in the amplified samples. From a procedural point of view, the viromes obtained by direct sequencing (without amplification step) provided a broader overview of both ss and dsDNA viral diversity. Nevertheless, our results suggest that the contextual use of random amplification of the same sample and long-read technology can improve the assessment of viral assemblages by reducing off-target reads. Full article
(This article belongs to the Section Bacterial Viruses)
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13 pages, 1980 KiB  
Article
Comprehensive Profiling of Alternative Splicing and Alternative Polyadenylation during Fruit Ripening in Watermelon (Citrullus lanatus)
by Yongtao Yu, Yuxiang Liufu, Yi Ren, Jie Zhang, Maoying Li, Shouwei Tian, Jinfang Wang, Shengjin Liao, Guoyi Gong, Haiying Zhang and Shaogui Guo
Int. J. Mol. Sci. 2023, 24(20), 15333; https://doi.org/10.3390/ijms242015333 - 18 Oct 2023
Cited by 5 | Viewed by 2128
Abstract
Fruit ripening is a highly complicated process that is accompanied by the formation of fruit quality. In recent years, a series of studies have demonstrated post-transcriptional control play important roles in fruit ripening and fruit quality formation. Till now, the post-transcriptional mechanisms for [...] Read more.
Fruit ripening is a highly complicated process that is accompanied by the formation of fruit quality. In recent years, a series of studies have demonstrated post-transcriptional control play important roles in fruit ripening and fruit quality formation. Till now, the post-transcriptional mechanisms for watermelon fruit ripening have not been comprehensively studied. In this study, we conducted PacBio single-molecule long-read sequencing to identify genome-wide alternative splicing (AS), alternative polyadenylation (APA) and long non-coding RNAs (lncRNAs) in watermelon fruit. In total, 6,921,295 error-corrected and mapped full-length non-chimeric (FLNC) reads were obtained. Notably, more than 42,285 distinct splicing isoforms were derived from 5,891,183 intron-containing full-length FLNC reads, including a large number of AS events associated with fruit ripening. In addition, we characterized 21,506 polyadenylation sites from 11,611 genes, 8703 of which have APA sites. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that fructose and mannose metabolism, starch and sucrose metabolism and carotenoid biosynthesis were both enriched in genes undergoing AS and APA. These results suggest that post-transcriptional regulation might potentially have a key role in regulation of fruit ripening in watermelon. Taken together, our comprehensive PacBio long-read sequencing results offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of watermelon fruit ripening. Full article
(This article belongs to the Special Issue Molecular Research of Tropical Fruit)
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13 pages, 1423 KiB  
Communication
Long-Read High-Throughput Sequencing (HTS) Revealed That the Sf-Rhabdovirus X+ Genome Contains a 3.7 kb Internal Duplication
by Hailun Ma, Trent J. Bosma and Arifa S. Khan
Viruses 2023, 15(10), 1998; https://doi.org/10.3390/v15101998 - 26 Sep 2023
Cited by 2 | Viewed by 1540
Abstract
We previously reported a novel rhabdovirus produced from the Spodoptera frugiperda Sf9 cell line, designated as Sf-rhabdovirus X+ since it contained a unique accessory gene X. The Sf-rhabdovirus X+ genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). [...] Read more.
We previously reported a novel rhabdovirus produced from the Spodoptera frugiperda Sf9 cell line, designated as Sf-rhabdovirus X+ since it contained a unique accessory gene X. The Sf-rhabdovirus X+ genome sequence was generated using Sanger sequencing and short-read high-throughput sequencing (HTS). In this study, we have used long-read HTS technologies, PacBio’s single-molecule real-time sequencing and Oxford’s Nanopore RNA direct sequencing, to analyze the parent Sf9 cell line transcriptome and the virus RNA produced from an X+ cell clone, respectively. A unique 3.7 kb duplication was identified in the L gene between nucleotide position 8523 and 8524, preceded by a GA dinucleotide insertion. This duplication contained a partial G gene, the complete X gene, and a partial L gene, which extended from nucleotide positions 4767–8523 in the X+ virus. Thus, the X+ genome length is 17,361 nucleotides, and we have re-designated the virus as Sf-rhabdovirus X+3.7. The 3.7 kb duplication was found in all Sf9 cell clones producing the X+ variant virus. Furthermore, the Sf-rhabdovirus X+3.7 genome was stable at passage 30, which was the highest passage tested. These results highlight the importance of combining short-read and long-read technologies for accurately sequencing virus genomes using HTS. Full article
(This article belongs to the Section Invertebrate Viruses)
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11 pages, 1715 KiB  
Communication
First Report of a Low-Frequency Mosaic Mutation in the Hydroxymethylbilane Synthase Gene Causing Acute Intermittent Porphyria
by Adrian Belosevic, Anna-Elisabeth Minder, Morgan Gueuning, Franziska van Breemen, Gian Andri Thun, Maja P. Mattle-Greminger, Stefan Meyer, Alessandra Baumer, Elisabeth I. Minder, Xiaoye Schneider-Yin and Jasmin Barman-Aksözen
Life 2023, 13(9), 1889; https://doi.org/10.3390/life13091889 - 10 Sep 2023
Cited by 1 | Viewed by 3079
Abstract
Acute porphyrias are a group of monogenetic inborn errors of heme biosynthesis, characterized by acute and potentially life-threatening neurovisceral attacks upon exposure to certain triggering factors. Biochemical analyses can determine the type of acute porphyria, and subsequent genetic analysis allows for the identification [...] Read more.
Acute porphyrias are a group of monogenetic inborn errors of heme biosynthesis, characterized by acute and potentially life-threatening neurovisceral attacks upon exposure to certain triggering factors. Biochemical analyses can determine the type of acute porphyria, and subsequent genetic analysis allows for the identification of pathogenic variants in the specific gene, which provides information for family counselling. In 2017, a male Swiss patient was diagnosed with an acute porphyria while suffering from an acute attack. The pattern of porphyrin metabolite excretion in urine, faeces, and plasma was typical for an acute intermittent porphyria (AIP), which is caused by inherited autosomal dominant mutations in the gene for hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway. However, the measurement of HMBS enzymatic activity in the erythrocytes was within the normal range and Sanger sequencing of the HMBS gene failed to detect any pathogenic variants. To explore the molecular basis of the apparent AIP in this patient, we performed third-generation long-read single-molecule sequencing (nanopore sequencing) on a PCR product spanning the entire HMBS gene, including the intronic sequences. We identified a known pathogenic variant, c.77G>A, p.(Arg26His), in exon 3 at an allelic frequency of ~22% in the patient’s blood. The absence of the pathogenic variant in the DNA of the parents and the results of additional confirmatory studies supported the presence of a de novo mosaic mutation. To our knowledge, such a mutation has not been previously described in any acute porphyria. Therefore, de novo mosaic mutations should be considered as potential causes of acute porphyrias when no pathogenic genetic variant can be identified through routine molecular diagnostics. Full article
(This article belongs to the Special Issue Heme Metabolism and Porphyria)
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17 pages, 5884 KiB  
Article
SMRT Sequencing Technology Was Used to Construct the Batocera horsfieldi (Hope) Transcriptome and Reveal Its Features
by Xinju Wei, Danping Xu, Zhiqian Liu, Quanwei Liu and Zhihang Zhuo
Insects 2023, 14(7), 625; https://doi.org/10.3390/insects14070625 - 11 Jul 2023
Cited by 5 | Viewed by 1821
Abstract
Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae) is an important forest pest in China that mainly infests timber and economic forests. This pest primarily causes plant tissue to necrotize, rot, and eventually die by feeding on the woody parts of tree trunks. To gain a [...] Read more.
Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae) is an important forest pest in China that mainly infests timber and economic forests. This pest primarily causes plant tissue to necrotize, rot, and eventually die by feeding on the woody parts of tree trunks. To gain a deeper understanding of the genetic mechanism of B. horsfieldi, this study employed single-molecule real-time sequencing (SMRT) and Illumina RNA-seq technologies to conduct full-length transcriptome sequencing of the insect. Total RNA extracted from male and female adults was mixed and subjected to SMRT sequencing, generating a complete transcriptome. Transcriptome analysis, prediction of long non-coding RNA (lncRNA), coding sequences (CDs), analysis of simple sequence repeats (SSR), prediction of transcription factors, and functional annotation of transcripts were performed in this study. The collective 20,356,793 subreads (38.26 G, clean reads) were generated, including 432,091 circular consensus sequences and 395,851 full-length non-chimera reads. The full-length non-chimera reads (FLNC) were clustered and redundancies were removed, resulting in 39,912 consensus reads. SSR and ANGEL software v3.0 were used for predicting SSR and CDs. In addition, four tools were used for annotating 6058 lncRNAs, identifying 636 transcription factors. Furthermore, a total of 84,650 transcripts were functionally annotated in seven different databases. This is the first time that the full-length transcriptome of B. horsfieldi has been obtained using SMRT sequencing. This provides an important foundation for investigating the gene regulation underlying the interaction between B. horsfieldi and its host plants through gene editing in the future and provides a scientific basis for the prevention and control of B. horsfieldi. Full article
(This article belongs to the Section Insect Molecular Biology and Genomics)
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21 pages, 2742 KiB  
Article
Assessing the Resilience of Machine Learning Classification Algorithms on SARS-CoV-2 Genome Sequences Generated with Long-Read Specific Errors
by Bikram Sahoo, Sarwan Ali, Pin-Yu Chen, Murray Patterson and Alexander Zelikovsky
Biomolecules 2023, 13(6), 934; https://doi.org/10.3390/biom13060934 - 2 Jun 2023
Viewed by 2473
Abstract
The emergence of third-generation single-molecule sequencing (TGS) technology has revolutionized the generation of long reads, which are essential for genome assembly and have been widely employed in sequencing the SARS-CoV-2 virus during the COVID-19 pandemic. Although long-read sequencing has been crucial in understanding [...] Read more.
The emergence of third-generation single-molecule sequencing (TGS) technology has revolutionized the generation of long reads, which are essential for genome assembly and have been widely employed in sequencing the SARS-CoV-2 virus during the COVID-19 pandemic. Although long-read sequencing has been crucial in understanding the evolution and transmission of the virus, the high error rate associated with these reads can lead to inadequate genome assembly and downstream biological interpretation. In this study, we evaluate the accuracy and robustness of machine learning (ML) models using six different embedding techniques on SARS-CoV-2 error-incorporated genome sequences. Our analysis includes two types of error-incorporated genome sequences: those generated using simulation tools to emulate error profiles of long-read sequencing platforms and those generated by introducing random errors. We show that the spaced k-mers embedding method achieves high accuracy in classifying error-free SARS-CoV-2 genome sequences, and the spaced k-mers and weighted k-mers embedding methods are highly accurate in predicting error-incorporated sequences. The fixed-length vectors generated by these methods contribute to the high accuracy achieved. Our study provides valuable insights for researchers to effectively evaluate ML models and gain a better understanding of the approach for accurate identification of critical SARS-CoV-2 genome sequences. Full article
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