DNA Sequence Research Articles

The Somatic Mosaicism across Human Tissues Network.

From fertilization onwards, the cells of the human body acquire variations in their DNA sequence, known as somatic mutations. These postzygotic mutations arise from intrinsic errors in DNA replication and repair, as well as from exposure to mutagens. Somatic mutations have been implicated in some diseases, but a fundamental understanding of the frequency, type and patterns of mutations across healthy human tissues has been limited. This is primarily due to the small proportion of cells harbouring specific somatic variants within an individual, making them more challenging to detect than inherited variants. Here we describe the Somatic Mosaicism across Human Tissues Network, which aims to create a reference catalogue of somatic mutations and their clonal patterns across 19 different tissue sites from 150 non-diseased donors and develop new technologies and computational tools to detect somatic mutations and assess their phenotypic consequences, including clonal expansions. This strategy enables a comprehensive examination of the mutational landscape across the human body, and provides a comparison baseline for somatic mutation in diseases. This will lead to a deep understanding of somatic mutations and clonal expansions across the lifespan, as well as their roles in health, inageing and, by comparison, in diseases.

GenoPath: a pipeline to infer tumor clone composition, mutational history, and metastatic cell migration events from tumor DNA sequencing data

DNA sequencing technologies are widely used to study tumor evolution within a cancer patient. However, analyses require various computational methods, including those to infer clone sequences (genotypes of cancer cell populations), clone frequencies within each tumor sample, clone phylogeny, mutational tree, dynamics of mutational signatures, and metastatic cell migration events. Therefore, we developed GenoPath, a streamlined pipeline of existing tools to perform tumor evolution analysis. We also developed and added tools to visualize results to assist interpretation and derive biological insights. We have illustrated GenoPath’s utility through a case study of tumor evolution using metastatic prostate cancer data. By reducing computational barriers, GenoPath broadens access to tumor evolution analysis. The software is available at https://github.com/SayakaMiura/GP.

Diet of Mainland Leopard Cat Prionailurus bengalensis within and outside a Protected Area in Arunachal Pradesh, India

Abstract The Mainland Leopard Cat (MLC) has a wide global distribution but information on its diet is restricted to a few locations. We collected 376 scats from Eaglenest Wildlife Sanctuary (EWS) and surrounding areas outside the Protected Area (PA) in Arunachal Pradesh, India, from 2014 to 2016. We assigned scats to MLC using felid-specific primers and DNA sequencing. Eighty-nine scats were used for diet estimation (PA: n = 45; outside PA: n = 44). We examined segregated prey items from scats under a microscope. Rodent teeth were identified to the family level. Rodents dominated the diet of MLC within and outside the PA, occurring in 96 % of all scats analysed and belonged to Family Muridae, Cricetidae and Sciuridae, with Muridae being the most common. The major difference in the diet of MLC within and outside the PA was in the consumption of the Sikkim Vole (Cricetidae) which occurred in a higher percentage of scats found within the PA. This difference was largely due to the higher elevations of scat locations within the PA. Our study emphasises the role of the MLC as a major predator of rodents.

First Report of Colletotrichum siamense Causing Anthracnose on Myrciaria dubia in Brazil.

Camu-camu (Myrciaria dubia (Kunth) McVaugh) is an Amazonian fruit with high economic potential due to its reported health-promoting properties, including elevated antioxidant and anti-inflammatory compound levels. Since 2018, anthracnose-like lesions, starting as small dark spots and coalescing into large irregular or circular lesions, were observed in up to 60% of camu-camu plants in an experimental field located in the city of Boa Vista, Roraima, Brazil (2°52'20.7″ N; 60°42'44.2″ W) and in a natural population in Rorainópolis, Roraima, Brazil (0°54'128″ N,60°36'226″ W). Fifty symptomatic leaves were collected from 10 plants. Small (5×5 mm) lesion tissue fragments were surface disinfected in 70% ethanol (1 min), 2.5% sodium hypochlorite (1 min), rinsed in sterile water, and placed on water agar. After 4 days at 25°C, mycelium was transferred to potato dextrose agar (PDA). After 7 days, 10 morphologically similar isolates were obtained, showing grayish-white colonies with orange spore masses. Conidia were hyaline, unicellular, cylindrical, smooth-walled, straight with rounded apices, measuring 15.3 ± 0.8 × 6.95 ± 1.4 μm (n = 30). Appressoria were brown, irregular or ovoid, measuring 6.4 ± 1.5 × 4.9 ± 1.2 μm (n = 30). Single-spore cultures were deposited in the EMBRAPA Roraima Microorganism Culture Collection. Four representative isolates (MTG07, MTG08 from Boa Vista; DXV01, DXV02 from Rorainópolis) were selected for phylogenetic and pathogenicity analysis. Total DNA was extracted and portions of actin (ACT), calmodulin (CAL), chitin synthase 1 (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin 2 (TUB2) genes were amplified (Weir et al. 2012) and Sanger sequenced (GenBank accession numbers ACT: PV463490-PV463493; CAL: PV463498-PV463501; CHS-1: PV055675-PV055678; GAPDH: PV430301-PV430304; TUB2: PV463494-PV463497). Sequences showed 96 to 99% identity with sequences of Colletotrichum siamense published in GenBank. Phylogenetic Bayesian inference analysis based on a combined data set showed that the isolates clustered with the ex-holotype specimen of C. siamense (ICMP 18578) with high support (posterior probability = 0.98). To confirm pathogenicity, five seedlings of camu-camu per isolate were sprayed with 106 conidia/mL spore suspension (15 mL/plant). Control seedlings were sprayed with sterile water. All seedlings were covered with plastic bags 24 h after inoculation and maintained at 27°C in a greenhouse with a 12-h photoperiod. After 6 days, inoculated leaves exhibited small dark brown spots with yellow halo, which enlarged and coalesced into regular or irregular brown necrotic lesions. The control plants showed no symptoms. The pathogenicity test was performed twice with similar results. The fungus was successfully reisolated from the inoculated leaves and its identify was confirmed by cultural morphology and DNA sequence. C. siamense is a known anthracnose agent on diverse hosts (Weir et al. 2012), but in camu-camu, only C. gloeosporioides, C. aeschynomenes, C. tropicale, and C. theobromicola (Perez et al., 2006; Matos et al., 2020; Sachet et al., 2024) have been reported. To our knowledge, this is the first report of C. siamense causing anthracnose in camu-camu. The identification of the pathogen enables future research on management strategies, as this pathogen can cause serious damage to camu-camu production in the region.

Investigation of LBX1, TIMP2, GPR126 and CHD7 Gene Polymorphisms in Adolescent Idiopathic Scoliosis Patients.

Study DesignProspective genetic cohort study.ObjectiveAdolescent idiopathic scoliosis (AIS) is a common spinal disorder affecting individuals aged 10-18 years without other underlying health conditions. This study aimed to examine the potential etiologic association between AIS and polymorphisms in the LBX1 (rs11190870, rs625039, rs11598564), TIMP2 (rs8179090), GPR126 (rs6570507), and CHD7 (rs121434341) genes in Turkish patients. Additionally, the relationships of these polymorphisms with sex, age, age at diagnosis, and Cobb angle were evaluated.MethodsThe study included 301 individuals: 201 patients with AIS (aged 10-18 years, Cobb angle ≥10°, no genetic disorders or related diseases), and 100 healthy controls (aged 10-18 years, no scoliosis diagnosis). The study analyzed rs625039, rs11598564, rs6570507, rs121434341, rs11190870, and rs8179090 polymorphisms in patients with AIS and controls using RT-PCR, confirmed the SNP regions through DNA sequencing, and performed statistical analysis.ResultsIn this study, the rs11190870 polymorphism of the LBX1 gene demonstrated a statistically significant difference between patients with AIS and the control group (P < .001), but no significance was observed for the other polymorphisms analyzed. Sex-based analysis revealed a significant association for the LBX1 rs11598564 polymorphism, with a higher frequency observed in females (P = .029); no significant differences were identified for the other polymorphisms in terms of sex. The rs8179090 and rs121434341 polymorphisms, previously associated with AIS in other populations, showed no statistically significant association in the present study cohort.ConclusionThe LBX1 gene rs11190870 polymorphism was found to be associated with AIS in Turkish patients.

Sources of uncertainty in DNA metabarcoding of whole communities: Implications for its use in biomonitoring

Abstract Advancements have been made in the use of DNA‐based methods for the detection of single species. However, the routine application of DNA‐based methods to monitor whole communities using a metabarcoding approach and derive ecosystem status continues to be limited. We undertook a structured experiment to assess factors influencing the precision and accuracy of molecular methods for freshwater macroinvertebrate community assessment. Macroinvertebrates were sorted, identified and counted from kick‐net samples using standard morphometric protocols, then reconstituted. These bulk specimen samples were then homogenised. From each bulk sample, aliquots of the homogenate were distributed among seven laboratories across Europe for DNA extraction, PCR amplification, library preparation and sequencing. Additionally, each laboratory was provided with a DNA extract from the bulk homogenate to allow for amplification through to sequencing, in order to assess the influence of DNA extraction. Hierarchical nested analyses were used to identify sources of uncertainty in the data and explore factors influencing the returned community and metrics of ecosystem quality. Furthermore, comparison with morphological analysis enabled us to benchmark molecular to traditional processing approaches. Metrics derived by morphological and DNA‐based methods differed substantially. The strongest methodological influences on variance in metrics derived from DNA‐based methods were attributed to sequencing (read count per sample) and laboratory (strong variation within samples). Furthermore, the performance of the DNA‐based method varied among samples, most likely due to differences in the composition of the invertebrate community. Logistic regression indicated that detectability did not vary among taxa, but that taxa occurring in low abundance (less than five individuals in our samples) were more likely to be missed via metabarcoding. Our findings highlight sources of uncertainty influencing DNA‐based methods and, particularly, the variability of results generated from individual laboratories. Our findings underline the need for clear guidelines, standardisation and quality assurance schemes.

Epigenome Engineering Using dCas Systems for Biomedical Applications and Biotechnology: Current Achievements, Opportunities and Challenges

Epigenome engineering, particularly utilizing CRISPR/dCas-based systems, is a powerful strategy to modulate gene expression and genome functioning without altering the DNA sequence. In this review we summarized current achievements and prospects in dCas-mediated epigenome editing, primarily focusing on its applications in biomedicine, but also providing a wider context for its applications in biotechnology. The diversity of CRISPR/dCas architectures is outlined, recent innovations in the design of epigenetic editors and delivery methods are highlighted, and the therapeutic potential across a wide range of diseases, including hereditary, neurodegenerative, and metabolic disorders, is examined. Opportunities for the application of dCas-based tools in animal, agricultural, and industrial biotechnology are also discussed. Despite substantial progress, challenges, such as delivery efficiency, specificity, stability of induced epigenetic modifications, and clinical translation, are emphasized. Future directions aimed at enhancing the efficacy, safety, and practical applicability of epigenome engineering technologies are proposed.

Get ready for short tandem repeats analysis using long reads-the challenges and the state of the art

Short tandem repeats (STRs) are repetitive DNA sequences that contribute to genetic diversity and play a significant role in disease susceptibility. The human genome contains approximately 1.5 million STR loci, collectively covering around 3% of the total sequence. Certain repeat expansions can significantly impact cellular function by altering protein synthesis, impairing DNA repair, and leading to neurodegenerative and neuromuscular diseases. Traditional short-read sequencing struggles to accurately characterize STRs due to its limited read length, which limits the ability to resolve repeat expansions, increases mapping errors, and reduces sensitivity for detecting large insertions or interruptions. This review examines how long-read sequencing technologies, particularly Oxford Nanopore and PacBio, overcome these limitations by enabling direct sequencing of full STR regions with improved accuracy. We discuss challenges in sequencing, bioinformatics workflows, and the latest computational tools for STR detection. Additionally, we highlight the strengths and limitations of different methods, providing deeper insight into the future of STR genotyping.

Incidence, Symptomology, Morpho-molecular Characterization of Agroathelia rolfsii (Sclerotium rolfsii Sacc.): The Causal Agent of Basal Rot in Chabaud Carnation

Background: Basal rot or sclerotium rot is one of the most serious and deadly disease in wide range of crops, including carnations. In carnations, it causes significant damage and economic loss. Methods: The Agroathelia rolfsii, basionym: Sclerotium rolfsii Sacc., the causal agent of basal rot in Chabaud carnation was ioslated from diseased plant parts collected from the Horticultural Research Farm, Assam Agricultural University, Jorhat, in 2023-2024 consequently, the study on the disease incidence (%), symptoms and morphomolecular characterization of the pathogen was carried out. Result: The incidence of basal rot was recorded the maximum in April (25.78%). The symptoms first appeared in the basal region, just above the ground, which gradually progressed upward over time. Colonizing white mycelia were conspicuous, producing white smooth fruiting bodies that turned brown, resembling mustard seed grains. The disease caused constriction at the infected point, resulting in decaying of the tissue and collapse of the plant eventually. The culture exhibited compact, whitish, cottony mycelia with sclerotia grown over time. Additionally, the DNA sequence of fungal isolate was submitted to NCBI, with an accession No. PP 203038. The fungal isolate had 92.99% similarity with A. rolfsii and a phylogenetic tree was constructed according to its nearest species with a bootstrap value.

Molecular Profiling of Selected Aspergillus Species Parasitizing Stored Wheat Seeds in the Silos of Tikrit City, Iraq

This study aimed to isolate and identify fungal species.AspergillusFrom wheat seeds stored in silos in Tikrit city, Salah al-Din Governorate, to assess the risks of fungal contamination on the safety of stored crops. Samples were collected from two local wheat seed varieties (with and without sorghum), and fungi were isolated from them using culture techniques on medium.PDA. Fungal isolates were identified based on morphological characteristics (colony color, texture, conidiophore and conidia shape), in addition to molecular identification using DNA sequencing of the ITS region of rRNA, after amplification using primers ITS1 and ITS4. BLAST analysis results showed a high match between the local isolates and the isolates registered in the GenBank database, where the following accession numbers were registered: A. fumigatus (PV363322), A. terreus (PV363323), A. niger (PV363324), and A. flavus (PV363325). Genetic analysis results also showed relative differences among some isolates, indicating potential genetic diversity within the local environment. These results demonstrate that stored wheat seeds are susceptible to contamination by fungal species known to produce dangerous mycotoxins, posing an environmental and health threat. Together, the results of molecular and phenotypic identification confirm the accuracy of isolate classification and its role in assessing fungal risks in storage silos.

Cutaneous Paraconiothyrium cyclothyrioides infection in a dog with insulinoma

ABSTRACTAn 8‐year‐old spayed female Chihuahua dog previously diagnosed with insulinoma was evaluated for a nonhealing wound on the right tarsal area. Cutaneous phaeohyphomycosis was diagnosed via histology. A pan‐fungal polymerase chain reaction test with DNA sequencing identified the causative agent as Paraconiothyrium cyclothyrioides, an environmental fungus and emerging pathogen rarely reported in humans and other animals. We describe the diagnostic workup utilised in this case and compare the advantages and pitfalls of using routine histology, histochemical special stains and molecular testing in cases of mycotic dermatitis.

A fuzzy sequencer for rapid DNA fragment counting and genotyping.

High-throughput sequencing technologies generate a vast number of DNA sequence reads simultaneously, which are subsequently analysed using the information contained within these fragmented reads. The assessment of sequencing technology relies on information efficiency, which measures the amount of information entropy produced per sequencing reaction cycle. Here we propose a fuzzy sequencing strategy that exhibits information efficiency more than twice that of currently prevailing cyclic reversible terminator sequencing methods. To validate our approach, we develop a fully functional and high-throughput fuzzy sequencer. This sequencer implements an efficient fluorogenic sequencing-by-synthesis chemistry and we test it across various application scenarios, including copy-number variation detection, non-invasive prenatal testing, transcriptome profiling, mutation genotyping and metagenomic profiling. Our findings demonstrate that the fuzzy sequencing strategy outperforms existing methods in terms of information efficiency and delivers accurate resequencing results with faster turnaround times.

Improved Visualization Method of DNA Sequences and its Application in Phylogenetic Analysis.

With a large number of species' genomes assembled, sequence comparison has become an effective method for further studying biological classification and evolution. Traditional sequence alignment relies on predefined scoring functions, but it is computationally intensive and lacks molecular justification for scoring the differences between sequences. Therefore, we have developed a graphical representation method for DNA sequences to facilitate better sequence comparison and evolutionary analysis. In this article, we introduce a novel method for representing DNA sequences using three-dimensional (3D) graphics. This method possesses two significant properties: (1) the graphical representation is acyclic; (2) each DNA sequence maintains a bijective relationship with its graphical representation. Leveraging this proposed visualization method, we computed the corresponding ALE index for any DNA sequence by converting it into an L/L matrix and constructed a 12-dimensional feature vector. The feasibility of our proposed method has been validated through the construction of phylogenetic trees in four test sets: terrestrial vertebrates, hantavirus, fish and Japanese encephalitis virus.

FGFR2 Genetic Variant C.1019A &gt; G p.(Tyr340Cys) in a Fetus with Pfeiffer Type II Syndrome Diagnosed by a Combination of 3D Ultrasound Phenotyping and DNA Sequencing

Abstract A 38-year-old woman was referred at 20 weeks' gestation for multiple fetal anomalies detected on routine ultrasound screening. Initial findings included dolichocephaly, ventriculomegaly, craniofacial abnormalities, and limb anomalies, which led to the suspicion of a skeletal dysplasia. While microarray analysis was normal, rapid fetal exome sequencing identified a de novo pathogenic variant in the FGFR2 gene (c.1019A &gt; G p.(Tyr340Cys)). Initially classified as a variant of uncertain significance due to initial ultrasound suspicion, targeted three and four dimensional ultrasound phenotyping revealed characteristic features of Pfeiffer syndrome type 2, including cloverleaf skull, proptosis, hypertelorism, broad thumbs and big toes, and radial-humeral synostosis. This case demonstrates the crucial interplay between advanced genetic testing and detailed ultrasound assessment in prenatal diagnosis. Accurate interpretation of genetic variants requires meticulous ultrasound phenotyping in a multidisciplinary setting, particularly for complex and rare conditions such as Pfeiffer syndrome type 2.

Size and Chemical Environment Control Nanopore Geometry in 2D MoS2: From Irregular to Triangular Defects.

Defects in 2D transition metal dichalcogenides (TMDs), such as molybdenum disulfide (MoS2), can modulate their optoelectronic and membrane properties. Increased structural complexity and a quasi-2D nature complicate the study of extended defects in MoS2. To address this knowledge gap, coordination-dependent atomic fingerprints are advanced for undercoordinated atoms in TMDs, enabling the cataloging of nanopore isomers in MoS2. Combining the introduced fingerprints with extensive density functional theory calculations of etching energies, stochastic kinetic Monte Carlo simulations of defect formation, and chemical graph theory for distinguishing nanopore shapes, predicts the most probable nanopores in MoS2. A range of size-dependent topologies are revealed from elongated to perfectly triangular, where smaller defects are irregular, while larger ones are more symmetric, exhibiting qualitative agreement with experiments. Moving toward a sulfur-rich chemical environment slows down the growth of larger pores and makes them perfectly triangular, providing an experimental route to control nanopore synthesis. The size-dependent structural order in MoS2 nanopores elucidated here will enable precise control over the defect shape and size distribution in the material for various application areas, including seawater desalination, gas separations, DNA sequencing, and optoelectronic devices.

Hb Lepore Rochester-MN, a novel βδβ double crossover hemoglobin variant.

Hb Lepore Rochester-MN, a novel βδβ double crossover hemoglobin variant.

Development of an allele-specific quantitative polymerase chain reaction assay for differentiating the RLB 106 strain from the wild-type viruses of Varicellovirus bovinealpha1.

Development of an allele-specific quantitative polymerase chain reaction assay for differentiating the RLB 106 strain from the wild-type viruses of Varicellovirus bovinealpha1.

Broadening the Nicotiana benthamiana research toolbox through the generation of dicer-like mutants using CRISPR/Cas9 approaches.

Broadening the Nicotiana benthamiana research toolbox through the generation of dicer-like mutants using CRISPR/Cas9 approaches.

Diagnosing Burkitt Lymphoma in Sub-Saharan Africa by Sequencing of Circulating Tumor DNA: A Comparative Microcosting Study.

Diagnosing Burkitt Lymphoma in Sub-Saharan Africa by Sequencing of Circulating Tumor DNA: A Comparative Microcosting Study.

Miocene marine transgression and climate variation drove diversification of subtropical Proceratophrys frogs in the Araucaria Forest

Abstract The Proceratophrys bigibbosa species group has a disjunct distribution in South America, with four out of five species inhabiting the Araucaria Forest. In this study, we investigated whether cycles of expansions/retractions of the Araucaria Forest or marine transgressions leading to the formation of the Paranean Sea may have contributed to the divergence of these frogs. It was inferred through DNA sequences from three mitochondrial and three nuclear genes across 82 individuals. Genetic divergence was assessed with haplotype networks, while demographic trends were explored using extended Bayesian skyline plots (EBSP). An environmental principal component analysis (ePCA) was also implemented to evaluate the degree of similarity of climatic spaces occupied by each species. Our phylogenetic analyses positioned P. palustris as the sister-species to P. brauni, forming a sister-clade to P. bigibbosa and an undescribed species (Proceratophrys sp.). In addition, we propose P. kaingang as a junior synonym of P. palustris. Our coalescent species tree showed divergence-time estimates ranging between 7.67 and 3.51 Mya, indicating that a marine transgression played a key role in this group diversification. Our findings highlight the combined role of Miocene marine transgressions and climatic variation as drivers of diversification in the Proceratophrys bigibbosa group.