Search Results (762)

Background/Objectives: 16S ribosomal RNA sequencing has, for several years, been the main means of identifying bacterial and archaeal species. Low-throughput Sanger sequencing is often used for the detection and identification of microbial species, but this technique has several limitations. The use of high-throughput sequencers may be a good alternative to improve patient identification, especially for polyclonal infections and management. Kraken 2 and KrakenUniq are free, high-throughput tools providing a very rapid and accurate classification for metagenomic analyses. However, Kraken 2 can present false-positive results relative to KrakenUniq, which can be limiting in hospital settings requiring high levels of accuracy. The aim of this study was to establish an alternative next-generation sequencing technique to replace Sanger sequencing and to confirm that KrakenUniq is an excellent analysis tool that does not present false results relative to Kraken 2. Methods: DNA was extracted from reference bacterial samples for Laboratory Quality Controls (QCMDs) and the V2-V3 and V3-V4 regions of the 16S ribosomal gene were amplified. Amplified products were sequenced with the Illumina 16S Metagenomic Sequencing protocol with minor modifications to adapt and sequence an Illumina 16S library with a small 500-cycle nano-flow cell. The raw files (Fastq) were analyzed on a commercial Smartgene platform for comparison with Kraken 2 and KrakenUniq results. KrakenUniq was used with a standard bacterial database and with the 16S-specific Silva138, RDP11.5, and Greengenes 13.5 databases. Results: Seven of the eight (87.5%) QCMDs were correctly sequenced and identified by Sanger sequencing. The remaining QCMD, QCMD6, could not be identified through Sanger sequencing. All QCMDs were correctly sequenced and identified by MiSeq with the commercial Smartgene analysis platform. QCMD6 contained two bacteria, Acinetobacter and Klebsiella. KrakenUniq identification results were identical to those of Smartgene, whereas Kraken 2 yielded 25% false-positive results. Conclusions: If Sanger identification fails, MiSeq with a small nano-flow cell is a very good alternative for the identification of bacterial species. KrakenUniq is a free, fast, and easy-to-use tool for identifying and classifying bacterial infections. Full article

Apricots are affected by many abiotic and biotic factors that could negatively impact their vitality and yield, leading to branch and tree dieback. Knowledge of the microbiome composition is key to choosing the optimal measurement strategy. The effect of the two different growing systems, i.e., organic (ORG) and integrated pest management (IPM), on the apricot fungal microbiome was studied. The inner bark was used to isolate DNA, and the present fungi were analyzed using a metagenomics high-throughput sequencing (HTS) profiling approach of the data obtained based on the Illumina sequencing of the ITS1-ITS2 amplicons of the 18S rRNA gene. Of the 20 analyzed samples, Ascomycota was the dominant phylum, and Dothiomycetes was the most abundant. Basidiomycota was the less frequent, with Tremellomycetes being the predominant within this phylum. PCA analysis showed the complete separation of the samples obtained from the orchards grown under the ORG and IPM systems. Cladosporia, Alternaria, Aureobasidium, and Visniacozyma were detected in all samples, but they dominated the IPM samples. Filobasiadiales were recognized as an indicator species for ORG management, while Caliciales, Lecanorales, Lichinales, Mycosphaerellales, Myriangiales, Phacidiales, Teloschistales, and Thelebolales were identified as indicator species for IPM management. Based on the order and genus levels, a significantly higher fungal microbiome richness was detected in the ORG samples. This could be connected to the environmentally beneficial growing system applied in the orchard, but it is impossible to assess the risk of trunk disease development or premature apricot tree decline. Full article

Microorganisms are crucial for the liquor brewing process and substantially impact liquor flavour and quality; therefore, understanding microbial succession is necessary. Most studies use a single-method approach and fail to provide an in-depth analysis. We aimed to combine traditional culture method with high-throughput sequencing (HTS) to identify the microbial diversity and succession in Luzhou-flavour fermentation. HTS revealed 932 bacterial and 980 fungal operational taxonomic units. 16S rDNA, 26S D1/D2 rDNA, and ITS v4/v5 isolated and identified 256 bacterial and 130 yeast strains. Population succession analysis showed that the dominant populations were yeasts, Lactobacillus, and Bacillus (early stage), and yeasts and Lactobacillus (late stage). Lactobacillus, Pichia, Bacillus, and Candida were abundant among all three layers of fermented grains. However, C. ethanolica, Saccharomycetes sp., and an unidentified Saccharomyces cerevisiae were more abundant in the lower layer than in the middle and upper layers, while L. parabuchneri, Oceanobacillus oncorhynchi, and Thermoactinomyces sp. were present only in the lower layer. Correlations among enzyme activity, volatile production, and dominant microbes during fermentation indicated that P. fermentans, L. suebicus, L. acetotolerans, P. kudriavzevii, P. exigua, and B. tequilensis were significantly affected during brewing. Our results lay a foundation for elucidating the microbial fermentation mechanism of Luzhou-flavour liquor and will assist in improving traditional liquor brewing quality and efficiency. Full article

Acute myeloid leukemia (AML), the most common acute leukemia among adults, poses significant therapeutic challenges due to diagnostic limitations and the frequent development of treatment resistance. While genomics-based approaches have advanced, DNA aberrations do not always reflect the expression levels of genes and proteins, which are more tightly connected to disease phenotypes. Recently, the role of the gut microbiota in AML has gained increasing attention. AML patients often exhibit gut microbiota dysbiosis, which is linked to disease progression and heightened infection risk. Mounting evidence indicates that gut microbiota metabolism influences hematopoiesis and immune function via the “gut-bone marrow axis,” with microbiota composition and diversity significantly affecting treatment outcomes and prognosis. High-throughput sequencing and metabolomics have identified correlations between gut microbiota composition and its metabolic products with AML clinical characteristics, paving the way for new biomarkers in diagnosis and prognosis. Additionally, treatments such as fecal microbiota transplantation (FMT) show promise in enhancing chemotherapy efficacy and improving patient outcomes. This review highlights recent advances in understanding the role of the gut microbiota in AML and explores new perspectives for its diagnosis and treatment. Full article

Studying the effects of environmental factors on microbial community assemblies is crucial for understanding microbial biodiversity and ecosystem processes. Although numerous studies have explored the spatial patterns of microbial communities in surface soils, bacterial community distributions in subsurface layers remain poorly understood. We investigated multiple community metrics of soil bacteria in arid and semi-arid grasslands in China, and the V4 region of 16S rDNA was analyzed using soil property measurements, fluorescent PCR, and high-throughput sequencing techniques. Specifically, copiotrophic taxa dominate the topsoil, whereas oligotrophic taxa are prevalent in nutrient-limited subsoil. Bacterial diversity decreases from the topsoil to subsoil, and bacterial distribution and ecological community composition exhibit a strong dependence on environmental factors. Moreover, microbial interaction networks demonstrated a progressive simplification with increasing soil depth: topsoil communities displayed higher modularity and a greater prevalence of positive interactions, whereas subsoil networks were significantly less complex. Null model analyses evidenced assembly mechanisms: deterministic processes (particularly homogeneous selection) dominated the bacterial community assembly, but their influence weakened with depth, whereas stochastic processes (e.g., dispersal limitation) increased progressively from the topsoil to subsoil. The PLS-PM analysis demonstrated that the relative influence of abiotic factors (e.g., climatic conditions and nutrient availability), biotic factors (interspecific interactions), along with drift and dispersal limitations on fungal community assembly exhibited depth-dependent patterns. This study provides novel insights into the vertical stratification of bacterial community in arid and semi-arid grasslands, and advances our understanding of pedogenic process under climate change and microbial adaptive strategies in heterogeneous soil environments. Full article

Global agricultural intensification has exacerbated soil compaction and nitrogen (N) inefficiency, thereby threatening sustainable crop production. Sub-soiling, a tillage technique that fractures subsurface layers while preserving surface structure, offers potential solutions by modifying soil physical properties and enhancing microbial-mediated N cycling. This study investigated the effects of subsoiling depth (0, 20, and 40 cm) on soil microbial communities and N transformations in a semi-arid maize system in China. The results demonstrated that subsoiling to a depth of 40 cm (D2) significantly enhanced the retention of nitrate-N and ammonium-N, which correlated with improved soil porosity and microbial activity. High-throughput 16S rDNA sequencing revealed subsoiling depth-driven reorganization of microbial communities, with D2 increasing the abundance of Proteobacteria (+11%) and ammonia-oxidizing archaea (Nitrososphaeraceae, +19.9%) while suppressing denitrifiers (nosZ gene: −41.4%). Co-occurrence networks indicated greater complexity in microbial interactions under subsoiling, driven by altered aeration and carbon redistribution. Functional gene analysis highlighted a shift from denitrification to nitrification-mineralization coupling, with D2 boosting maize yield by 9.8%. These findings elucidate how subsoiling depth modulates microbiome assembly to enhance N retention, providing a mechanistic basis for optimizing tillage practices in semi-arid agroecosystems. Full article

The development of molecules that interact with G-quadruplex (G4) sequences requires effective evaluation methods. Several techniques are currently available, including nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS), fluorescence using FRET-melting, G4-fluorescent intercalator displacement assay (G4-FID) and affinity chromatography. Among these, CD spectroscopy is gaining prominence due to its lower material requirements, faster experimentation and quicker data processing. However, conventional CD methods have limitations, such as higher sample volume required and the inability to handle high-throughput analysis efficiently. The use of synchrotron radiation in high-throughput analysis methods (HT-SRCD) has further advanced the investigation of small-molecule interactions with DNA G4 structures in the presence of various monovalent cations. HT-SRCD offers the capability to analyze multiple samples simultaneously, overcoming the limitations of conventional CD methods. To validate this approach, three biologically relevant G4 sequences—HTelo1, G3T3 and T95-2T—were investigated. Their interactions with a library of small tetrazole-based molecules, synthesized via a four-component Ugi reaction, and with a peptide sequence deriving from RHAU helicases (Rhau25), were evaluated. The results demonstrate that this method not only effectively discriminates between different ligands but also provides valuable insights into the selectivity and the modes of interaction of these ligands with the G4 sequences. Full article

Grapevine (Vitis vinifera L.) cultivation is an important agricultural sector worldwide. Its expansion into new areas, like Kazakhstan, brings significant phytosanitary risks. Viroids, such as grapevine yellow speckle viroid 1 (GYSVd-1) and hop stunt viroid (HSVd), are RNA pathogens that threaten vineyard productivity. They can cause a progressive decline through latent infections. Traditional diagnostic methods are usually targeted and therefore not suitable for thorough surveillance. In contrast, modern high-throughput sequencing (HTS) methods often face challenges due to their high costs and complicated sample preparation, such as ribosomal RNA (rRNA) depletion. This study introduces a simplified diagnostic workflow that overcomes these barriers. We utilized the latest Oxford Nanopore V14 cDNA chemistry, which is designed to prevent internal priming, by substituting a targeted oligo(dT)VN priming strategy to facilitate the sequencing of non-polyadenylated viroids from total RNA extracts, completely bypassing the rRNA depletion step and use of random oligonucleotides for c DNA synthesis. This method effectively detects and identifies both GYSVd-1 and HSVd. This workflow significantly reduces the time, cost, and complexity of HTS-based diagnostics. It provides a powerful and scalable tool for establishing strong genomic surveillance and phytosanitary certification programs, which are essential for supporting the growing viticulture industry in Kazakhstan. Full article

Tumor heterogeneity encompasses genetic, epigenetic, and phenotypic diversity, impacting treatment response and resistance. Spatial heterogeneity occurs both inter- and intra-lesionally, while temporal heterogeneity results from clonal evolution. High-throughput technologies like next-generation sequencing (NGS) enhance tumor characterization, but conventional biopsies still do not adequately capture genetic heterogeneity. Liquid biopsy (LBx), analyzing circulating tumor DNA (ctDNA), provides a minimally invasive alternative, offering real-time tumor evolution insights and identifying resistance mutations overlooked by tissue biopsies. This study evaluates the capability of LBx to capture tumor heterogeneity by comparing genetic profiles from multiple metastatic lesions and LBx samples. Eight patients from the Augsburger Longitudinal Plasma Study with various types of cancer provided 56 postmortem tissue samples, which were compared against pre-mortem LBx-derived circulating-free DNA sequenced by NGS. Tissue analyses revealed significant mutational diversity (4–12 mutations per patient, VAFs: 1.5–71.4%), with distinct intra- and inter-lesional heterogeneity. LBx identified 51 variants (4–17 per patient, VAFs: 0.2–31.1%), which overlapped with mutations from the tissue samples by 33–92%. Notably, 22 tissue variants were absent in LBx, whereas 18 LBx-exclusive variants were detected (VAFs: 0.2–2.8%). LBx effectively captures tumor heterogeneity, but should be used in conjunction with tissue biopsies for comprehensive genetic profiling. Full article

Papillomaviruses (PVs) are double-stranded DNA viruses known to induce a variety of epithelial lesions in dogs, ranging from benign hyperplasia to malignancies. In regions of rich biodiversity such as the Western Amazon, data on the circulation and genetic composition of canine papillomaviruses (CPVs) remain scarce. This study investigated CPV types present in oral and cutaneous papillomatous lesions in domiciled dogs from Acre and Rondônia States, Brazil. Sixty-one dogs with macroscopically consistent lesions were clinically evaluated, and tissue samples were collected for histopathological examination and PCR targeting the L1 gene. Among these, 37% were histologically diagnosed as squamous papillomas or fibropapillomas, and 49.2% (30/61) tested positive for papillomavirus DNA. Sequencing of the L1 gene revealed that most positive samples belonged to CPV1 (Lambdapapillomavirus 2), while one case was identified as CPV8 (Chipapillomavirus 3). Complete genomes of three CPV1 strains were obtained via high-throughput sequencing and showed high identity with CPV1 strains from other Brazilian regions. Phylogenetic analysis confirmed close genetic relationships among isolates across distinct geographic areas. These findings demonstrate the circulation of genetically conserved CPVs in the Amazon and reinforce the value of molecular and histopathological approaches for the accurate diagnosis and surveillance of viral diseases in domestic dogs, especially in ecologically complex regions. Full article

Environmental DNA (eDNA) analysis has emerged as a transformative tool in environmental monitoring, enabling non-invasive detection of species and microbial communities across diverse ecosystems. This study systematically reviews the role of bioinformation technology in eDNA analysis, focusing on methodologies and applications across air, soil, groundwater, sediment, and aquatic environments. Advances in molecular biology, high-throughput sequencing, bioinformatics tools, and field-deployable detection systems have significantly improved eDNA detection sensitivity, allowing for early identification of invasive species, monitoring ecosystem health, and tracking pollutant degradation processes. Airborne eDNA monitoring has demonstrated potential for assessing microbial shifts due to air pollution and tracking pathogen transmission. In terrestrial environments, eDNA facilitates soil and groundwater pollution assessments and enhances understanding of biodegradation processes. In aquatic ecosystems, eDNA serves as a powerful tool for biodiversity assessment, invasive species monitoring, and wastewater-based epidemiology. Despite its growing applicability, challenges remain, including DNA degradation, contamination risks, and standardization of sampling protocols. Future research should focus on integrating eDNA data with remote sensing, machine learning, and ecological modeling to enhance predictive environmental monitoring frameworks. As technological advancements continue, eDNA-based approaches are poised to revolutionize environmental assessment, conservation strategies, and public health surveillance. Full article

Background/Objectives: The gene pool of the Apennine wolf is affected by admixture with domestic variants due to anthropogenic hybridisation with dogs. Genetic monitoring at the population level involves assessing the extent of admixture in single individuals, ranging from pure wolves to recent hybrids or wolf backcrosses, through the analysis of nuclear and mitochondrial DNA (mtDNA) markers. Although individually non-diagnostic, mtDNA is nevertheless essential for completing the final diagnosis of genetic admixture. Typically, the identification of wolf mtDNA haplotypes is carried out via sequencing of coding genes and non-coding DNA stretches. Our objective was to develop a fast real-time PCR assay to detect the mtDNA haplotypes that occur exclusively in the Apennine wolf population, as a valuable alternative to the demanding sequence-based typing. Methods: We validated a qualitative duplex real-time PCR that exploits the combined presence of diagnostic point mutations in two mtDNA segments, the NDH-4 gene and the control region, and is performed in a single-tube step through TaqMan-MGB chemistry. The aim was to detect mtDNA multi-fragment haplotypes that are exclusive to the Apennine wolf, bypassing sequencing. Results: Basic validation of 149 field samples, consisting of pure Apennine wolves, dogs, wolf × dog hybrids, and Dinaric wolves, showed that the assay is highly specific and sensitive, with genomic DNA amounts as low as 10⁻⁵ ng still producing positive results. It also proved high repeatability and reproducibility, thereby enabling reliable high-throughput testing. Conclusions: The results indicate that the assay presented here provides a valuable alternative method to the time- and cost-consuming sequencing procedure to reliably diagnose the maternal lineage of the still-threatened Apennine wolf, and it covers a wide range of applications, from scientific research to conservation, diagnostics, and forensics. Full article

Efficient and consistent DNA extraction is crucial for genotyping but often hindered by the limitations of traditional manual processes, which are labour-intensive, error-prone, and costly. We introduce a semi-automated, robotic-assisted DNA extraction (RoboCTAB) tailored for large-scale plant genotyping, leveraging advanced yet affordable liquid-handling robotic systems. The protocol/workflow integrates a CTAB extraction protocol specifically adapted for a robotic liquid-handling system, making it compatible with high-throughput genotyping techniques such as SNP genotyping and sequencing. Various plant parts (leaves, roots, manual seed chip) were explored as the source material for DNA extractions, with the aim of identifying the tissue best suited for collection on a large scale. Young roots (radicle) proved the easiest to harvest at scale, while the harvest of leaves and seed chips were more laborious and error-prone. DNA yield and quality from both leaves and roots (but not seed chips) were similar and sufficient for downstream analysis. Interestingly, root tissue could still be extracted from imbibed seeds, even if the seeds failed to germinate, thus proving useful for DNA extraction. Cost analysis indicates significant savings in labour costs, highlighting the approach’s suitability for large-scale projects. Quality assessments demonstrate that the robotic process yields high-quality DNA, maintaining integrity for downstream applications. This semi-automated DNA extraction system represents a scalable, reliable solution for large-scale genotyping that is accessible to many users who cannot implement highly sophisticated and costly systems as are known to exist in large multinational seed companies. RoboCTAB, a low-cost, optimized method for high-throughput DNA extraction, minimizes the risk of cross-contamination. RoboCTAB is capable of processing up to four 96-well plates (384 samples) simultaneously in a single run, improving cost-efficiency and providing seamless integration with laboratory workflows, potentially setting new standards for efficiency and quality in DNA processing and sequencing at scale. Full article

The consumption and farming of insects are gaining global attention as sustainable alternatives to conventional protein sources. Industrial processing of insects into powders or pastes complicates species identification, raising concerns about product authenticity, food safety, and potential fraud. In Western countries, particularly in Europe, the sector is expanding under a stringent regulatory framework, especially regarding rearing substrates, which hinders economic development. This study aimed to assess the species authenticity of commercial insect-based food and feed products and detect the presence of animal-derived DNA from unauthorized substrates. A total of 119 samples (pure insect meals and processed products) were collected from various origins. Species-specific real-time PCR assays targeted Tenebrio molitor, Hermetia illucens, Alphitobius diaperinus, Acheta domesticus, Bombyx mori, and Gryllodes sigillatus, alongside assays for ruminant, porcine, and poultry DNA. High-throughput sequencing (HTS) using metabarcoding confirmed and broadened species detection. Most samples contained the declared species; however, cases of mislabeling, substitution, and cross-contamination were observed. A few insect meals contained animal DNA which could suggest potential use of prohibited substrates. These findings highlight the urgent need for standardized authentication methods and improved transparency to ensure regulatory compliance, consumer trust, and sustainable development of the insect-based sector. Full article

Circulating cell-free DNA (cfDNA) is an important biomarker for various cancer types, enabling a non-invasive testing approach. However, pre-analytical variables, including sample collection, tube type, processing conditions, and extraction methods, can significantly impact the yield, integrity, and overall quality of cfDNA. This study presents a comprehensive analytical validation of a magnetic bead-based, high-throughput cfDNA extraction system, with a focus on assessing its efficiency, reproducibility, and compatibility with downstream molecular applications. The validation was performed using a range of sample types: synthetic cfDNA spiked into DNA-free plasma, multi-analyte ctDNA plasma controls, Seraseq ctDNA reference material in a plasma-like matrix, extraction specificity controls, residual clinical specimen from patients, and samples from healthy individuals stored at room temperature or 4 °C for up to 48 h to assess stability. Extracted cfDNA was analyzed for concentration, percentage, and fragment size, using the Agilent TapeStation. Variant detection was evaluated using a next-generation sequencing (NGS) assay on the Seraseq ctDNA reference material. The results demonstrated high cfDNA recovery rates, consistent fragment size distribution (predominantly mononucleosomal and dinucleosomal), minimal genomic DNA (gDNA) contamination, and strong concordance between detected and expected variants in reference materials. The workflow also showed robust performance under different study parameters, variable sample conditions, including sample stability and integrity. Together, these findings confirm the efficiency and reliability of the evaluated cfDNA extraction system and underscore the importance of standardized pre-analytical workflows for the successful implementation of liquid biopsy for early cancer detection, therapeutic monitoring, and improved patient outcomes. Full article