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PubMed2024

High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing.

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput, comparatively to the actual sequencing costs. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS™ 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit excelled across all performance parameters. We further used the nanoliter dispensing system I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample. This enhanced efficiency and cost-effectiveness will enable researchers to undertake studies with greater sample sizes and diversity, thereby providing a richer, more detailed view of microbial communities and their dynamics.

Metagenome↗

PubMed2026

Liquid fermentation and an adapted cetyltrimethylammonium bromide (CTAB) method enable sequencing-grade DNA extraction from Phanerodontia australis.

High-quality genomic DNA extraction from basidiomycete fungi remains challenging due to polysaccharide co-purification and nuclease-mediated DNA degradation. We systematically compared 22 experimental conditions combining five DNA extraction methods (one Dellaporta-based method, three CTAB-based variants, and one commercial kit) with five mycelium preparation techniques (scalpel scraping, fungal suspension, silica bead homogenization, liquid fermentation, and liquid nitrogen treatment) for Phanerodontia australis (BRM62389). DNA quality was assessed by 1% agarose gel electrophoresis, NanoDrop spectrophotometry, and Qubit fluorimetry. The optimal protocol combined liquid-fermentation mycelium with an adapted CTAB method, yielding Qubit-quantified concentrations of 34.2 and 62.6 ng/µL (samples S21 and S22), 260/280 ratios of 2.06 and 2.05, 260/230 ratios of 1.88 and 1.85, and no detectable DNA degradation by agarose gel electrophoresis. This protocol enabled whole-genome sequencing, yielding a 37.62 Mb assembly with 98.5% completeness as assessed by BUSCO v5 (basidiomycota_odb10 lineage dataset, n = 1,764). Liquid fermentation is associated with the production of younger, actively growing hyphae with reduced cell wall thickness, and the adapted CTAB method effectively removed residual polysaccharides. This protocol provides a reliable, cost-effective approach for obtaining sequencing-grade DNA from P. australis.

Basidiomycota↗

PubMed2026

Comparative performance of portable DNA extraction protocols and bioinformatics workflows for rapid detection of gram-negative bacteria and antimicrobial resistance using Oxford Nanopore sequencing.

Oxford Nanopore Technology (ONT) enables rapid, portable pathogen identification and antimicrobial resistance (AMR) detection, but the reliability of downstream genomic analyses is highly dependent on DNA extraction quality, particularly in resource-limited settings. This study comparatively evaluated four portable bacterial DNA extraction protocols derived from three commercial kits to determine their impact on nanopore sequencing performance, bioinformatics workflow completion, and field deployability. Six gram-negative bacterial isolates (Escherichia coli, n = 4; Pseudomonas sp., n = 1; and Salmonella sp., n = 1) were processed using four extraction protocols: SwiftX DNA, SwiftX DNA with proteinase K (ProtK), SwiftX ParaBact, and NucleoSpin Microbial. Twenty-four resulting DNA extracts were sequenced on a single multiplexed MinION R10.4.1 flow cell. Sequencing data were analyzed using validated Galaxy-based generic and species-specific pipelines. Workflow completion was defined as successful progression through quality control, assembly, virulence, plasmid, and AMR detection modules. DNA purity varied substantially by extraction protocol and was strongly associated with successful workflow completion (Kruskal-Wallis, P = 0.0006). Accordingly, NucleoSpin Microbial achieved 100% workflow completion, and SwiftX ParaBact achieved 83%, while both SwiftX DNA-based protocols failed to complete full workflows. Importantly, key AMR genes required to classify isolates as multidrug-resistant were consistently detected using both NucleoSpin Microbial and SwiftX ParaBact extractions. However, NucleoSpin Microbial assemblies showed significantly higher contiguity and enabled a broader, more complete detection of virulence factors, pathogenicity islands, plasmid replicons, and accessory AMR genes, reflecting enhanced genomic resolution.IMPORTANCERapid whole-genome sequencing is increasingly used to detect antimicrobial resistance and guide public health responses, but its reliability depends strongly on how bacterial DNA is extracted. In this study, we have shown that DNA extraction method choice has a major impact on Oxford Nanopore sequencing performance across clinically relevant gram-negative bacteria. While silica column-based extraction maximized genomic completeness and analytical depth, paramagnetic bead-based reverse purification offered superior portability with sufficient resolution for frontline AMR surveillance. These findings highlight a practical trade-off between field deployability and high-resolution genomic characterization in low-resource settings.

DNA extraction↗

PubMed2026

Comparative evaluation of three high-molecular-weight DNA extraction kits for Oxford Nanopore sequencing of Clostridioides difficile and Clostridium perfringens.

UNLABELLED: Clostridioides difficile and Clostridium perfringens are Gram-positive, spore-forming anaerobic pathogens affecting humans and animals, for which genomic data have been mainly generated using short-read or hybrid sequencing approaches. In this study, we evaluated three commercial non-bead-beating DNA extraction kits designed for high-molecular-weight DNA recovery for Oxford Nanopore long-read whole-genome sequencing of two C. difficile and two C. perfringens strains, including one reference strain and one clinical or environmental isolate per species. Based on sequencing performance and kit ease of use, one kit was selected for additional sequencing of plasmid-carrying strains of both species. All three kits allowed correct identification of sequence types, toxin-encoding genes, and antimicrobial resistance determinants, confirming their suitability for clinical and epidemiological applications. However, the BT MasterPure Kit provided the highest DNA concentrations, longest fragment sizes, and superior read lengths and N50 values, particularly for C. difficile, achieving >100× coverage and enabling reliable circularization of chromosomes and plasmids, including a C. difficile metronidazole resistance plasmid and C. perfringens plasmids carrying toxin and antibiotic resistance genes. The other kits produced slightly lower DNA yields, resulting in shorter reads and reduced genome coverage for C. difficile, highlighting the challenge of extracting high-quality DNA from Gram-positive, spore-forming bacteria. Overall, this study provides practical guidance for selecting DNA extraction protocols optimized for Oxford Nanopore sequencing of C. difficile and C. perfringens, supporting high-quality genome assemblies and plasmid characterization and facilitating the routine genomic surveillance of clinically relevant spore-forming pathogens. IMPORTANCE: High-quality genomic data are essential for accurate characterization of Clostridioides difficile and Clostridium perfringens, two clinically and epidemiologically important Gram-positive, spore-forming pathogens. However, long-read sequencing performance can be strongly influenced by the choice of DNA extraction method, particularly for organisms with robust cell walls, where commonly used methods can lead to fragmented DNA. In this work, DNA of four strains was extracted using three commercial high-molecular-weight DNA extraction kits and sequenced using Oxford Nanopore Technologies. The best-performing kit was also evaluated using three additional strains known to harbor plasmids in order to assess its plasmid recovery efficiency. The results demonstrated successful plasmid recovery, circularization, and characterization. DNA extraction protocols optimized for Oxford Nanopore sequencing enable the rapid and cost-effective characterization of C. difficile and C. perfringens for genomic surveillance or outbreak investigations.

Clostridioides difficile↗

PubMed2026

DNA Extraction Optimisation for Minute Land Snails of Vertigo Müller, 1773 (Gastropoda: Vertiginidae): A Comparative Evaluation of Six Methods, Including a Non-Destructive Shell-Preserving Protocol.

No systematic comparison of DNA extraction strategies exists for minute Vertiginidae (shell height < 3 mm), a group posing a dual analytical challenge: extremely low tissue input and co-purified PCR-inhibitory mucus. For legally protected species, an additional requirement to preserve the shell voucher further constrains available protocols. Using Vertigo antivertigo as the model species, we compared six approaches applied to specimens preserved in 96% ethanol (n = 10 per method): two HotSHOT alkaline-lysis protocols (destructive and non-destructive shell-preserving variants), a modified CTAB protocol supplemented with PVP-40 and DTT, and three commercial silica-column kits (GeneJET Genomic, DNeasy Blood & Tissue, QIAamp DNA Micro). DNA yields were quantified by QuantiFluor fluorometry, and PCR performance was subsequently assessed across four loci (COI barcode, COI mini-barcode, ITS1, ITS2). DNeasy Blood & Tissue produced the highest fluorometric concentrations; QIAamp DNA Micro and CTAB + PVP-40 gave intermediate values. The shell-preserving HotSHOT variant yielded lower concentrations but improved A260/230 ratios. BSA and trehalose supplementation increased PCR success in inhibition-prone HotSHOT extracts from 70% to 100%. ITS1 Sanger sequencing of three Vertigo species listed in Annex II of the EU Habitats Directive, all extracted with the shell-preserving protocol, confirmed species-level identification (99.8%-100% BLASTn identity; mean Phred Q > 51). The shell-preserving non-destructive HotSHOT protocol yields sequenceable DNA from protected Vertiginidae while retaining the morphological voucher, making it the preferred option for conservation-genetic monitoring. The practical decision framework documented here-integrating voucher preservation, amplification robustness and per-sample cost-has broad applicability to other minute terrestrial gastropods processed in large-scale biodiversity surveys.

Habitats Directive↗

PubMed2026

Benchmarking DNA extraction protocols across use cases for culture-independent Nanopore metagenomics.

Oxford Nanopore Technologies (ONT) sequencing offers several advantages for metagenomics, including long reads, rapid turnaround, low upfront cost, scalability and portability. However, for ONT metagenomics, DNA yield, quality and integrity are important considerations when selecting an extraction method. Many metagenomic extraction methods use harsh lysis conditions to extract a wide range of species and provide an accurate community composition, but these conditions can compromise DNA fragment length. Therefore, extraction methods for ONT metagenomics must balance DNA shearing and recovery with representative community lysis. We systematically evaluated DNA extraction methods for ONT metagenomic sequencing using a use case-oriented framework. Among nearly 50 extraction methods screened, 7 were selected for detailed comparison based on suitability for metagenomics, variation in methodology, availability, cost and processing time: Norgen BioTek Corp's Stool DNA Isolation (NG), Zymo Research's ZymoBIOMICS Quick-DNA HMW MagBead (ZMG), Qiagen's DNeasy Blood and Tissue (QBT), Macherey-Nagel's NucleoMag DNA Microbiome (MN), Zymo Research's ZymoBIOMICS DNA Mini Prep (ZMI), Qiagen's DNeasy PowerSoil/QIAamp PowerFecal Pro (PS) and Qiagen's QIAamp Fast DNA Stool Mini (QIA). Methods were tested using Zymo Research's ZymoBIOMICS Microbial Community Standard (MCS), a matrix-free mock community with known composition. DNA extracts were sequenced on an ONT PromethION using the Rapid Barcoding Kit, except QIA due to insufficient DNA yield. Metrics for the method, DNA extracts, sequencing and genomes were evaluated, revealing trade-offs between methods. The two magnetic bead methods, MN and ZMG, produced the highest mean read length N50 values (13.9 and 16.5 kb, respectively) but showed apparent community compositions skewed towards Gram-negative bacteria. In contrast, ZMI and PS maintained a community composition close to expected, with reduced mean read length N50 values (4.5 vs. 7.5 kb). Performance across various metrics is presented in the context of the following use cases: maximizing genome coverage and assembly completeness, preserving composition accuracy, targeting specific species and limiting required resources (equipment, time or budget). The metrics and use case considerations presented offer practical guidance for informed selection of DNA extraction methods for ONT metagenomics. For accurate community composition, ZMI or PS are recommended, while PS and ZMG perform best at maximizing genome coverage and assembly completeness. NG and QBT may be the most economical options, though performance trade-offs were observed. Finally, PS may be the preferred method for time-sensitive diagnostic or field applications.

Metagenomics↗

PubMed2026

Selective Extraction of Genomic DNA From Animal Tissues Using a Hydrophobic Magnetic Ionic Liquid.

The development of green and efficient methods for genomic DNA extraction from animal tissues is crucial for molecular diagnostics, food traceability, and genetic research. Conventional methods often involve toxic reagents, multiple centrifugation steps, and are time-consuming. In this study, a hydrophobic magnetic ionic liquid (MIL), N-octyl-4-dimethylaminopyridinium hexafluorophosphate MIL ([C8DMAP][PF6]‑Ni MIL), was synthesized and applied for the selective extraction of genomic DNA from various animal tissues. The material exhibited strong paramagnetic behavior, high thermal stability, and excellent hydrophobicity, enabling rapid phase separation under an external magnetic field. A mechanical shaking-assisted extraction method was developed, and key parameters including temperature, time, shaking speed, and [C8DMAP][PF6]-Ni MIL dosage were systematically optimized. The method demonstrated high selectivity for DNA over proteins, RNA, and amino acids, with a maximum recovery rate of 78.06 ± 1.91%. Compared to a commercial DNA extraction kit, the [C8DMAP][PF6]-Ni MIL-based approach provided higher yields from several tissues, including mouse liver, brain, and rabbit lung. Furthermore, the [C8DMAP][PF6]-Ni MIL could be reused for at least six cycles while maintaining extraction efficiency. This work not only provides a high-performance material for DNA extraction, but also demonstrates a sustainable and easily retrievable liquid-phase separation strategy, offering a generalizable platform for complex sample pretreatment.

Animals↗

PubMed1976

Studies on the molecular species of DNA polymerase extracted from rat ascites hepatoma cells.

DNA polymerase [EC 2.7.7.7] activities present in hypotonic extract from rat ascites hepatoma AH130 cells were eluted in three separable peaks on DEAE-cellulose column chromatography. Peak I activity had an alkaline pH optimum, and was relatively resistant to SH-blocking reagents and salt concentration. These properties of DEAE peak I are typical of low molecular weight DNA polymerase. DEAE peak II and peak III activities possessed properties corresponding to high molecular weight (6-8 S) polymerase; they showed maximal activity at neutral pH, and were sensitive to SH-blocking reagents and salt. No low molecular weight polymerase activity was released from DEAE peak II or peak III by salt treatment, though partial conversion from DEAE peak II to peak III was observed on the same treatment.

Animals↗

PubMed2026

EST-SSR based genetic polymorphism among Lablab (Lablab purpureus L. Sweet) accessions contrasting for drought stress at seedling stage.

Lablab is a multipurpose and the most drought-tolerant (DT) crop compared with its relatives. Despite its potential, Lablab is still an underutilized crop with a lack of improved varieties in many countries. The DT (D349, D147, HA4, D363, D352, D359, D348, D311, D55 and D250) and drought-susceptible (DS) (D271, D66, D106, D6, D26, D255, D28, D186, D95, and D258) accessions were earlier identified according to their morphological and biochemical responses to moisture stress at the seedling stage. These accessions were used to establish genetic polymorphism among the accessions contrasting for drought stress based on the Expressed Sequence Tag-Simple Sequence Repeats (EST-SSR) markers. The CTAB protocol was employed for the genomic DNA extraction. After DNA quality and quantity verification, the PCR was conducted using 16 EST-SSR primer pairs specific to the Lablab. The products were separated through the horizontal polyacrylamide gel electrophoresis (hPAGE). Discriminating ability of the markers and primers' efficiency were evaluated based on various genetic parameters. Principal Coordinate Analysis (PCoA) was performed to estimate the distance matrix among the population and among the accessions. While cluster analysis was processed to trace the genetic relationship among the accessions, dendrogram was constructed to decipher their genetic relationship. Analysis of Molecular Variance (AMOVA) was finally computed to quantify the diversity level and genetic relationship among the population, and among the accessions. A low polymorphism (GD = 0.19) was observed between the DT and DS accessions, likely due to limited discriminatory power of the EST-SSR markers. However, the PCoA, cluster analysis and AMOVA identified DT (D147, HA4, and D349) and DS (D106, D95, and D271) accessions as strongly contrasting populations under drought stress, with D147, HA4, D349, D363, D359, D352, and D348 further recommended as DT accessions. Given the low polymorphism observed, further validation using more informative molecular markers and advanced genomic approaches is recommended to improve the identification of drought-tolerance genes and related QTLs to support Lablab breeding programs.

Expressed Sequence Tags↗

PubMed2024

Optimized methods for the targeted surveillance of extended-spectrum beta-lactamase-producing Escherichia coli in human stool.

Understanding transmission pathways of important opportunistic, drug-resistant pathogens, such as extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, is essential to implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To link transmission of ESBL-producing E. coli (ESBL-EC) between two sources, single-nucleotide resolution of E. coli strains, as well as E. coli diversity within and between samples, is required. However, the microbiological methods to best track these pathogens are unclear. Here, we compared different steps in the microbiological workflow to determine the impact different pre-enrichment broths, pre-enrichment incubation times, selection in pre-enrichment, selective plating, and DNA extraction methods had on recovering ESBL-EC from human stool samples, with the aim to acquire high-quality DNA for sequencing and genomic epidemiology. We demonstrate that using a 4-h pre-enrichment in Buffered Peptone Water, plating on cefotaxime-supplemented MacConkey agar and extracting DNA using Lucigen MasterPure DNA Purification kit improves the recovery of ESBL-EC from human stool and produced high-quality DNA for whole-genome sequencing. We conclude that our optimized workflow can be applied for single-nucleotide variant analysis of an ESBL-EC from stool.IMPORTANCEDrug-resistant infections are increasingly difficult to treat with antibiotics. Preventing infections is thus highly beneficial. To do this, we need to understand how drug-resistant bacteria spread to take action to stop infection and transmission. This requires us to accurately trace these bacteria between different sources. In this study, we compared different laboratory methods to see which worked best for detecting extended-spectrum beta-lactamase (ESBL)-producing E. coli, a common cause of urinary tract or bloodstream infections, from human stool samples. We found that enriching stool in a nutrient broth for 4 h, then plating the bacterial suspension on antibiotic-selective MacConkey agar, and finally extracting DNA from the bacteria using a specific DNA purification kit resulted in improved recovery of ESBL E. coli and high-quality DNA. Sequencing multiple isolates from stool allowed us to distinguish unambiguously and at high resolution between different variants of ESBL E. coli present in stool.

Humans↗

PubMed2026

Comparative metagenomic analysis of microbial communities: unravelling microbial communities from the great Rann of Kachchh and coastal saltpans, Gujarat, India.

Hypersaline environments exhibit extreme physiochemical conditions yet support diverse microbial communities. These communities are not only ecologically important but also possess substantial potential for biotechnological exploitation. In this study, we employed a comparative metagenomic approach to assess microbial diversity using two distinct methodologies: (1) direct DNA extraction from raw sediment, and (2) DNA extraction following halophilic enrichment in selective media. Sediment samples were collected from multiple sites and pooled together within the Rann of Kachchh and close-by saltpans and were analysed using 16S rRNA sequencing coupled with bioinformatics pipelines. The results revealed pronounced differences in microbial community composition between the two approaches. Raw sediment samples exhibited significantly higher alpha diversity, with dominant taxa including Halobacterota, Cyanobacteria, and Desulfobacterota, with a substantial proportion of unclassified genera. In contrast, enriched samples were dominated by fast-growing, culturable genera such as Halobacterium, Alkalibacillus, and Candidatus haloredivivus. Principal Coordinate Analysis (PCoA) of beta diversity demonstrated distinct clustering between raw and enriched communities, even within samples from the same sites, underscoring the selective bias introduced by enrichment procedures. These findings emphasise that the methodological choice strongly influences the observed microbial diversity. The aim of this study was to compare microbial community composition in raw hypersaline sediments and enrichment cultures using metagenomic sequencing, to evaluate how enrichment selectively favours specific halophilic taxa. This comparative approach allows identification of the microbial groups that rapidly proliferate under controlled hypersaline conditions, thereby complementing direct environmental sequencing. By integrating both direct and enrichment-based metagenomic approaches, a more comprehensive understanding of microbial community structure in hypersaline environments can be achieved.

India↗

PubMed2024

Advancing responsible genomic analyses of ancient mollusc shells.

The analysis of the DNA entrapped in ancient shells of molluscs has the potential to shed light on the evolution and ecology of this very diverse phylum. Ancient genomics could help reconstruct the responses of molluscs to past climate change, pollution, and human subsistence practices at unprecedented temporal resolutions. Applications are however still in their infancy, partly due to our limited knowledge of DNA preservation in calcium carbonate shells and the need for optimized methods for responsible genomic data generation. To improve ancient shell genomic analyses, we applied high-throughput DNA sequencing to 27 Mytilus mussel shells dated to ~111-6500 years Before Present, and investigated the impact, on DNA recovery, of shell imaging, DNA extraction protocols and shell sub-sampling strategies. First, we detected no quantitative or qualitative deleterious effect of micro-computed tomography for recording shell 3D morphological information prior to sub-sampling. Then, we showed that double-digestion and bleach treatment of shell powder prior to silica-based DNA extraction improves shell DNA recovery, also suggesting that DNA is protected in preservation niches within ancient shells. Finally, all layers that compose Mytilus shells, i.e., the nacreous (aragonite) and prismatic (calcite) carbonate layers, with or without the outer organic layer (periostracum) proved to be valuable DNA reservoirs, with aragonite appearing as the best substrate for genomic analyses. Our work contributes to the understanding of long-term molecular preservation in biominerals and we anticipate that resulting recommendations will be helpful for future efficient and responsible genomic analyses of ancient mollusc shells.

Animals↗

PubMed1978

Transfection in pneumococcus: single-strand intermediates in the formation of infective centers.

Transfection has been found and characterized in pneumococcus. For replicating omega3 phage DNA extracted from infected cells, transfection was relatively efficient and rose linearly with DNA concentration and quadratically with time, according to T(T - 3.5) min(2). For mature DNA extracted from phage particles, transfection was hardly detectable below 1 mug/ml but increased about as the cube of the DNA concentration up to 100 mug/ml, and was still rising at concentrations over 200 mug/ml. The kinetics suggest a dependence on a mixed cubic function of the time of exposure of cells to mature DNA. Cell and phage DNAs competed with each other for transformation and transfection. Transfection was reduced much more strongly than transformation in cells that were deficient in the membrane-bound endonuclease required for conversion of donor duplex DNA to intracellular single strands; these data agree with the kinetic data in implying that independent entry of segments of two strands is necessary for transfection by replicating omega3 phage DNA and entry of at least three strands is necessary for transfection by mature DNA. To reconcile differing DNA concentration dependences of transfection and transformation with a common entry path, it was necessary to reexamine data on transformation and to recognize that this process continued to rise slowly through the concentration region usually described as "plateau." These results and the transfection data reflect multiple binding and nicking events that occurred on the cell surface before entry. Our conclusion is that transfection in pneumococcus occurs by association inside the cell of segments of single strands of phage DNA that have entered independently, creating gapped structures that need repair synthesis to create infective centers. Physical recombination is therefore automatically a prerequisite to transfection.

Bacteriophages↗

PubMed2025

HLA-Typing of Donor-Origin Cells Enriched From Urine Cell Culture of Kidney Transplanted Recipients.

The incomplete or lack of histocompatibility information constitutes a barrier for the early detection and management of de novo donor-specific antibodies (DSA). To improve the quantity and quality of DNA materials for HLA typing, we developed a non-invasive culture-based method, using DNA extracted from enriched donor-derived kidney stem cells (DKSC) selectively cultured from the urine of kidney transplant receipients (KTR) to allow high-resolution typing by next-generation sequencing. This prospective proof-of-concept study evaluated the feasibility and performance of this approach. DKSC were enriched from the urine of 60 KTRs. DNA extracted from culture-enriched DKSC showed significantly higher concentration and better quality than unbound cells, and with identical short tandem repeat (STR) and 100% concordance compared to that obtained from peripheral blood. Our results suggest that cultured-enriched DKSC are non-invasive and useful for determining HLA and other genes for KTRs where donor information is limited or lacking.

Humans↗

PubMed2026

Oxford Nanopore Sequencing of Clinical DNA for Identification and Comparative Genomic Analysis of Erysipelothrix piscisicarius.

The genus Erysipelothrix comprises facultative anaerobic, nonspore-forming, gram-positive bacteria that can cause skin infections and severe diseases such as septicemia and endocarditis in humans. Although E. rhusiopathiae is the primary pathogen, other species may also be involved, necessitating accurate identification. However, 16S rDNA sequencing lacks sufficient resolution to differentiate among Erysipelothrix species. In this study, we used Oxford Nanopore Technology (ONT) to directly sequence low-quality DNA extracted from heart valve tissue of a 66-year-old female patient with a fatal case of septicemia and aortic endocarditis. In contrast to 16S rDNA Illumina sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which incorrectly identified the pathogen as E. rhusiopathiae, direct sequencing via ONT precisely identified E. piscisicarius as the cause of infection. About 1.47 Mb genome was retrieved from nanopore direct sequencing. Within the E. piscisicarius genome, we detected genes associated with virulence. Phylogenetic analysis showed that our strain clustered with a human-derived E. piscisicarius strain from China and swine-derived strains from Brazil. In conclusion, this study demonstrated that ONT can be used to sequence low-quality DNA extracted directly from patient specimens, obtain a draft bacterial genome, and reliably distinguish between pathogenic species.

Aged↗

PubMed2026

Evaluating detection of Histophilus somni immunoglobulin-binding protein A DR2 Fic: A species-specific gene target for recombinase polymerase amplification relative to long-read sequencing of respiratory samples from feedlot calves.

Histophilosis is an important cause of morbidity and mortality as well as antimicrobial use in feedlot cattle across North America. Detection of Histophilus somni by culture is challenging, and there is no standardized tool for distinguishing isolates that carry virulence factors most likely to contribute to disease. The DR2 repeat of H. somni-associated virulence factor 'immunoglobulin-binding protein A' (ibpA DR2) harbors a Fic domain that mediates host cell cytotoxicity and is essential for histophilosis. For rapid detection of ibpA DR2 in extracted DNA, we developed a real-time recombinase polymerase amplification (RPA) assay with a runtime of 24 min at 39 °C. DNA from H. somni-RPA-positive respiratory swabs (n = 73) was screened for ibpA DR2 using the novel RPA assay and long-read metagenomic sequencing, as well as nanopore whole-genome sequencing (WGS) of H. somni isolated from the same samples. IbpA DR2 was identified in 71% and 70% of tested samples using RPA and WGS, respectively, and in ≤41% of samples using metagenomic sequencing. The likelihood of detection by RPA did not differ (OR 1.1, 95% CI (0.42, 2.9), P > 0.99) from WGS; however, agreement between these assays was only fair (κ = 0.31). Conversely, RPA (OR 3.4, 95% CI (1.6, 8.2)) and WGS (OR 8.0, 95% CI (2.4, 42)) were more likely (P < 0.001) to detect ibpA DR2 than metagenomic sequencing, likely reflecting limited coverage of H. somni by metagenomics. This study demonstrated that RPA and long-read WGS detected ibpA DR2 with similar frequencies in extracted DNA and H. somni isolates, respectively. Further testing of non-target isolates confirmed the analytical specificity of ibpA DR2 to H. somni. Further investigation of the diagnostic validity for RPA-based ibpA DR2 detection is required in a larger cohort of field samples, as a rapid screening tool for H. somni most likely to contribute to disease.

Animals↗

PubMed2025

Development of a multiplex PCR for detection of pathogenic Mycobacterium orygis in cattle tissues harboring tuberculous-like lesions.

Mycobacterium orygis, a recently defined member species of Mycobacterium tubercuolsis complex (MTBC), is emerging as a major threat to zoonotic tuberculosis control, especially in the Asian Subcontinent. The dearth of low-cost diagnostic assay to differentiate M. orygis from other members of the MTBC leads to unavailability of information about the actual burden of this species in human and animal population. In this study, we developed a multiplex PCR for distinguishing M. orygis from other MTBC based on two M. orygis-specific nonsynonymous point mutations in mbtG and fadD23 genes identified by comparative genome analysis. The specificity of the assay shows that a 434 bp IS1081 fragment was amplified from common MTBC species including M. orygis while 240 bp and 181 bp mbtG and fadD23 gene fragments were amplified only from M. orygis. No amplification was observed for nontuberculous Mycobacterium (NTM) and non-Mycobacterial pathogens. The multiplex PCR assay showed a detection limit of 32 pg of M. orygis DNA. Furthermore, a total of 85 tuberculous-like lesions in the different tissues of slaughtered cattle were tested for identification of the M. orygis, and the results showed IS1081, mbtG and fadD23 amplicons in three tissue DNA extracts confirming they contain M. orygis DNA. Also, a single IS1081 amplicon was amplified from one tissue sample signifying presence of DNA of any MTBC species other than M. orygis. An established TaqMan real time PCR assay targeting region of differences (RD) in M. orygis genome was carried out to validate the result of the assay. This showed 100 % accuracy of the in-house developed multiplex PCR.

Mycobacterium orygis↗

PubMed2024

Sex differences in MAGEL2 gene promoter methylation in high functioning autism - trends from a pilot study using nanopore Cas9 targeted long read sequencing.

BACKGROUND: MAGEL2 is an autism susceptibility gene whose deficiency has been associated with autism-related behaviors in animal models and in syndromic human autism spectrum disorders (ASDs) such as Schaaf-Yang syndrome, but has not been studied in the broader autism spectrum. Given the capabilities of long-read sequencing technologies, this pilot study used a targeted nanopore sequencing approach to simultaneously examine MAGEL2 DNA sequence and methylation in adults with high-functioning autism (HFA) compared to neurotypical controls (NC). METHODS: Using DNA extracted from peripheral blood, Cas9-targeted nanopore DNA sequencing was used to analyze MAGEL2, including its entire regulatory construct (chr15:23639316-23651466), for sequence variation and 5-methyl-cytosine (5mC) modification in a cohort of adults with HFA compared to sex- and age-matched NC. Given the known sex differences in ASD and MAGEL2 KO animal models, results were further analyzed by sex. RESULTS: 20 adults with HFA (10 males, 10 females) and 20 NC were included. While there were no overall differences in MAGEL2 DNA sequence and 5mC modification between HFA and NC, we found a significant difference in MAGEL2 gene promoter methylation between males and females with HFA and NC of both sexes, with HFA males tending to show hypomethylation in a 300 bp long differentially methylated region (chr15:23647640-23647939) around the MAGEL2 transcription start site. CONCLUSIONS: In this pilot study utilizing nanopore Cas9 targeted DNA sequencing, significant sex-specific differences in MAGEL2 gene promoter methylation were identified in male adults with HFA in comparison to control groups, suggesting the potential for sex-specific epigenetic differences. However, further replication in larger cohorts is required to validate these findings.

Humans↗