Fast, Flexible, Feasible: A Transparent Framework for Evaluating eDNA Workflow Trade-offs in Resource-Limited Settings

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Апрель 18, 2025

Abstract Environmental DNA (eDNA) analysis enables biodiversity monitoring by detecting organisms from trace genetic material, but high reagent costs, cold-chain logistics, and computational demands limit its broader use, particularly in resource-limited settings. To address these challenges improve accessibility, we systematically evaluated multiple workflow components, including four extraction methods, two primer sets, three Nanopore basecalling models, demultiplexing pipelines. Across 144 combinations tested an aquarium with 15 fish species, mapped trade-offs between cost, sensitivity, processing speed using a hierarchical Bayesian model, to assess where time resource savings are possible without compromising detection. Workflows the Qiagen Blood Tissue (BT) kit provided highest recovering all species of sampled community within 3–5 hours Oxford sequencing when paired MiFish-U set high-accuracy (HAC) basecalling. Chelex, alternative lower-cost method, required extended (>24 hours) reach comparable species-detection rates. DirectPCR QuickExtract offered field-friendly alternatives that achieved recovery ∼10–12 hours, though their cost-effectiveness varied. While MarVer1 was designed broaden vertebrate detection, it recovered same as MiFish-U, fewer total reads. Real-time trials (0–61 revealed high-efficiency workflows (BT + HAC) reached detection plateaus rapidly, indicating can be reduced sacrificing accuracy. The OBITools4 bioinformatics pipeline enabled automated discarded more reads than alternative, ONTbarcoder2.3, which retained low-abundance taxa at cost manual curation. Rather identifying single “best” workflow, this study provides transparent decision framework for prioritizing speed, sensitivity eDNA applications, supporting scalable, cost-effective monitoring,

Язык: Английский

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Validation of a comprehensive long-read sequencing platform for broad clinical genetic diagnosis

Frontiers in Genetics, Год журнала: 2025, Номер 16

Опубликована: Май 2, 2025

Though short read high-throughput sequencing, commonly known as Next-Generation Sequencing (NGS), has revolutionized genomics and genetic testing, there is no single test that can accurately detect nucleotide variants (SNVs), small insertions/deletions (indels), complex structural (SVs), repetitive genomic alterations, in genes with highly homologous pseudogenes. The implementation of a unified comprehensive technique simultaneously broad spectrum variation would substantially increase efficiency the diagnostic process. current study evaluated clinical utility long-read sequencing for diagnosis inherited conditions. Using Oxford Nanopore Technologies long nanopore we successfully developed validated clinically deployable integrated bioinformatics pipeline utilizes combination eight publicly available variant callers. A concordance assessment comparing calls from well-characterized, benchmarked sample called NA12878 National Institute Standards Technology (NIST) detected by our this sample, determined analytical sensitivity was 98.87% specificity exceeded 99.99%. We then pipeline’s ability to 167 relevant 72 samples. This set consisted 80 SNVs, 26 indels, 32 SVs, 29 repeat expansions, including 14 overall detection these 99.4% (95% CI: 99.7%–99.9%). Importantly, addition detecting variants, four cases, yielded valuable additional information support diagnoses could not have been established using short-read NGS alone. Our findings suggest successful identifying diverse alterations functions well basis patients suspected disease.

Язык: Английский

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Robot‐Aided Measurement of Insect Diversity on Vegetation Using Environmental DNA

Ecology and Evolution, Год журнала: 2025, Номер 15(5)

Опубликована: Май 1, 2025

ABSTRACT Traditional methods of biodiversity monitoring are often logistically challenging, time‐consuming, require experienced experts on species identification, and sometimes include destruction the targeted specimens. Here, we investigated a non‐invasive approach combining use drones environmental DNA (eDNA) to monitor insect vegetation. We aimed assess efficiency this novel method in capturing diversity comparing composition across different vegetation types (grassland, shrub forest) Switzerland. A commercial, off‐the‐shelf drone was equipped with specialised probe that autonomously swabbed collected eDNA. Then, samples were processed using rapid third‐generation Oxford Nanopore sequencing. The obtained data analysed for diversity, taxonomic richness, evenness community three habitat statistical techniques. Sequencing yielded 76 hexapod taxa, revealing an notable differences richness but not grassland, forest habitats. Our study demonstrates potential drone‐based sampling integrated eDNA nanopore sequencing monitoring, offering non‐destructive detecting occurrence plant surfaces. Integrating robotics technology provides promising solution fast, large‐scale, potentially improving conservation efforts ecosystem management.

Язык: Английский

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Morphological and Molecular Evidence of the Presence of the Ninespined Stickleback Pungitius pungitius (Linnaeus, 1758) Within Ukrainian Gasterosteids, Including New mtDNA Haplotypes—Which Issue for Fish Conservation in Ukraine?

Aquatic Conservation Marine and Freshwater Ecosystems, Год журнала: 2025, Номер 35(5)

Опубликована: Май 1, 2025

ABSTRACT The Gasterosteidae, a family of marine, brackish and fresh water fish, is represented in Ukraine by the genera Pungitius Gasterosteus . Among these, only species platygaster has hitherto been identified. However, our findings based on molecular cox1 DNA barcoding collected material have confirmed additional presence pungitius Dnipro River Ukraine. This long remained hidden from scientific scrutiny, due to its diminutive size, lack commercial significance morphological similarities with sister species. Our analysis allowed conclusive identification this elusive provided an insight into distribution ecological relevance within inland waters To examine compare haplotype diversity, we analysed data P. , aculeatus those available public repositories. demonstrate novel haplotypes region for all support theory not recent invasion region. valuable role advanced methodologies unravelling biodiversity aquatic ecosystems, particularly case overlooked often marginalized taxa. We also explore current worldwide regional conservation status species, contributing future studies population management.

Язык: Английский

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eProbe: Sampling of Environmental DNA within Tree Canopies with Drones

Environmental Science & Technology, Год журнала: 2024, Номер 58(37), С. 16410 - 16420

Опубликована: Сен. 5, 2024

Environmental DNA (eDNA) analysis is a powerful tool for studying biodiversity in forests and tree canopies. However, collecting representative eDNA samples from these high complex environments remains challenging. Traditional methods, such as surface swabbing or rolling, are labor-intensive require significant effort to achieve adequate coverage. This study proposes novel approach unmanned aerial vehicles (UAVs) collect within canopies by using technique. The method involves lowering probe hovering UAV into the canopy it descends ascends through branches leaves. To this, custom-designed robotic system was developed featuring winch collection. design of optimized, control logic reduce risk entanglement while ensuring sufficient interaction force facilitate transfer onto probe. effectiveness this demonstrated during XPRIZE Rainforest Semi-Finals 10 were collected rainforest canopy, total 152 molecular operational taxonomic units (MOTUs) identified metabarcoding. We further investigate how number interactions with vegetation, penetration depth, sampling duration influence concentration community composition samples.

Язык: Английский

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Primed and ready: nanopore metabarcoding can now recover highly accurate consensus barcodes that are generally indel-free

BMC Genomics, Год журнала: 2024, Номер 25(1)

Опубликована: Сен. 9, 2024

Язык: Английский

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DNA Barcoding for Species Identification of Moss-Dwelling Invertebrates: Performance of Nanopore Sequencing and Coverage in Reference Database

Diversity, Год журнала: 2024, Номер 16(4), С. 196 - 196

Опубликована: Март 25, 2024

In view of the current biodiversity crisis and our need to preserve improve ecosystem functioning, efficient means for characterizing monitoring are required. DNA barcoding, especially when coupled with new sequencing technologies, is a promising method that can, in principle, also be employed by taxonomic lay people. this study we compare performance barcoding third-generation technology, nanopore classical Sanger sequencing, based on sample invertebrates collected from moss pads bog Austria. We find pipeline generates barcodes at least as good generated MinION producing better results than Flongle flowcell. further while many arthropod taxa well covered international reference barcode database BOLD, clearly not case important like mites springtails, which hampers large-scale assessments. Based examples highlight factors might responsible ambiguous species identification BOLD how partly, solved.

Язык: Английский

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Scalable, Cost-Effective, and Decentralized DNA Barcoding with Oxford Nanopore Sequencing

Methods in molecular biology, Год журнала: 2024, Номер unknown, С. 223 - 238

Опубликована: Янв. 1, 2024

Язык: Английский

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Implementing high-throughput insect barcoding in microbiome studies: impact of non-destructive DNA extraction on microbiome reconstruction

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Апрель 30, 2024

Abstract Background Symbiotic relationships with diverse microorganisms are crucial for many aspects of insect biology. However, while our understanding taxonomic diversity and the distribution species in natural communities is limited, we know much less about their microbiota. In era rapid biodiversity declines, as researchers increasingly turn towards DNA-based monitoring, developing broadly implementing approaches high-throughput cost-effective characterization both insect-associated microbial essential. We need to verify whether such barcoding, a powerful tool identifying wild insects, would permit subsequent microbiota reconstruction these specimens. Methods High-throughput barcoding (“megabarcoding”) methods often rely on non-destructive obtaining template DNA PCR amplification by leaching out specimens using alkaline buffers HotSHOT. This study investigated impact HotSHOT abundance estimates reconstructed bacterial community profiles. addressed this question comparing quantitative 16S rRNA amplicon sequencing data HotSHOT-treated or untreated 16 representing six orders selected based expectation limited variation among individuals. Results find that 13 species, treatment significantly reduced estimates, corresponding an estimated 15-fold decrease amplifiable average. On other hand, pre-treatment had effect composition. The presence abundant bacteria known significant effects was not affected. observed changes low-abundance microbes, those close reliable detection threshold. Alpha beta analyses showed compositional differences only few species. Conclusion Our results indicate pre-treated remain suitable composition reconstruction, even if may be hard estimate. These can cost-effectively combine across communities. Thus, voucher obtained megabarcoding studies targeted at characterizing used microbiome characterizations. substantially aid speeding up accumulation knowledge microbiomes hyperdiverse

Язык: Английский

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Hotspot of Exotic Benthic Marine Invertebrates Discovered in the Tropical East Atlantic: DNA Barcoding Insights from the Bijagós Archipelago, Guinea-Bissau

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Июль 10, 2024

Abstract Aim This study aimed to explore and document putative exotic marine benthic invertebrate species in the Bijagós Archipelago, Guinea-Bissau, enhance understanding of biodiversity address extent introductions. Location The research was conducted a UNESCO Biosphere Reserve located Guinea-Bissau. Methods involved region’s first scuba-diving survey biodiversity. DNA barcoding employed assist identification species. Molecular phylogenetic analyses were with available barcodes ensure accurate taxonomic assignments, detect cryptic species, investigate phylogeography taxa. Results resulted discovery 28 new records for including octocorals, scleractinians, hydroids, bryozoans, barnacles, ascidians. Among these, seven documented time East Atlantic: Stragulum bicolor , Tubastraea tagusensis Nemalecium lighti Diphasia sp., Amathia alternata A. distans Symplegma rubra . revealed pervasive diversity within previously listed as exotic, suggesting that some, such hydroids Plumularia setacea Obelia geniculata Dynamena disticha are not due their restricted biogeographic distributions. Many other reported introduced present only few genetic lineages capable long-distance dispersal human activities. Main Conclusions highlights considerable gaps knowledge West African suggests substantial underestimation anthropogenic trade between Tropical Atlantic Americas, Indo-Pacific Africa. Detailed genomic necessary species’ biogeography adaptive traits. Our findings challenge current classifications underscore need improved monitoring management prevent spread non-native

Язык: Английский

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