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ABSTRACT This minireview will discuss the improvements in Oxford Nanopore (Oxford; sequencing technology that make the MinION a viable platform for microbial ecology studies. Specific issues being addressed are the increase in sequence accuracy from 65 to 96.5% during the last 5 years, the ability to obtain a quantifiable/predictive signal from the MinION with respect to target molecule abundance, simple-to-use GUI-based pathways for data analysis and the modest additional equipment needs for sequencing in the field. Coupling these recent improvements with the low capital costs for equipment and the reasonable per sample cost makes MinION sequencing an attractive option for virtually any laboratory. The Oxford Nanopore MinION is the preferred platform for studies in microbial ecology, because of its long-read capability, low cost and ease of use.

Antimicrobial resistance genes (ARGs) in aquatic environments pose a significant threat to public health and ecological balance. This study investigates the diversity of ARGs in Laguna Lake, Philippines, using nanopore-based metagenomics and specialized bioinformatics tools. Water samples were collected in September 2023 and March 2024 from various locations within the lake. These samples underwent DNA extraction, library preparation, and sequencing with the Oxford Nanopore MinION device. Reads were processed to remove low-quality sequences and subjected to taxonomic and ARG classification using various bioinformatic tools. Taxonomic analysis revealed that Proteobacteria [September (46.87%) and March (42.02%)], Actinobacteria (17.18 to 24.44%), and Firmicutes (3.93 to 8.94%) were the dominant Phyla, showing seasonal variations in their relative abundances. ARG analysis revealed multiple antibiotic types and subtypes in the lake, with multidrug resistance genes most prevalent. Notable differences in ARG types and read counts were observed between the two sampling periods. The occurrence of these genes per phylum was also identified. The study provides insights into the impact of environmental factors, including temperature and human activities, on the microbial community and ARG dissemination within the lake, warranting further investigation. Lastly, these findings underscore the need for advanced genomic techniques and bioinformatic tools to understand and mitigate the spread of antimicrobial resistance in aquatic ecosystems.  

Long read sequencing technologies provide an efficient approach to generating highly contiguous and informative assemblies. However, higher relative error rates can introduce frameshifts and premature stop codons that pseudogenize genes, hindering downstream analyses. We developed a software tool that detects gene-fragmenting errors in draft assemblies of small genomes through comparison with a curated set of reference genome sequences and raw read information. In our presented example, detected errors represent less than 0.05% of the genome, but when corrected reduced the rate of pseudogenes from 23.3 to 5.6% in example long read assemblies, comparable to the rate of pseudogenes in short read assemblies. We demonstrate that this software can detect assembly errors in long read assemblies generated from small genomes and correct them to de-fragment genes.

ABSTRACT A total of four strains of the ‘environmental superbug’ Pedobacter isolated from sludge produced at Norwegian drinking water treatment plants, were characterized by whole genome sequencing and antibiotic susceptibility assays. As with previous studies on members of this genus, we found that the isolates were multi-drug resistant, and that this resistance included clinically important beta-lactams, aminoglycosides and the fluoroquinolone ciprofloxacin. Using the minION sequencing platform (Oxford Nanopore Technologies) combined with HiSeq PE150 Illumina sequencing data, the four isolates were assembled into genomes of single contigs. Analysis of the genomes revealed potential genetic factors possibly underlying some of the specific resistances observed. Metallo-beta-lactamase activity was detected in one isolate, and the same isolate contained a putative metallo-betalactamase gene resembling pedo-2. Furthermore, several genes related to multidrug efflux systems were found using the resistance database CARD. Additionally, the present study extends our knowledge on the phylogeny of this genus, adding four new genomes to the existing 50. Whole genome sequencing of the environmental superbugs™ Pedobacter spp. provides insights on their multi-drug resistance.

Background The application of long-read sequencing using the Oxford Nanopore Technologies (ONT) MinION sequencer is getting more diverse in the medical field. Having a high sequencing error of ONT and limited throughput from a single MinION flowcell, however, limits its applicability for accurate variant detection. Medical exome sequencing (MES) targets clinically significant exon regions, allowing rapid and comprehensive screening of pathogenic variants. By applying MES with MinION sequencing, the technology can achieve a more uniform capture of the target regions, shorter turnaround time, and lower sequencing cost per sample. Method We introduced a cost-effective optimized workflow, ECNano, comprising a wet-lab protocol and bioinformatics analysis, for accurate variant detection at 4800 clinically important genes and regions using a single MinION flowcell. The ECNano wet-lab protocol was optimized to perform long-read target enrichment and ONT library preparation to stably generate high-quality MES data with adequate coverage. The subsequent variant-calling workflow, Clair-ensemble, adopted a fast RNN-based variant caller, Clair, and was optimized for target enrichment data. To evaluate its performance and practicality, ECNano was tested on both reference DNA samples and patient samples. Results ECNano achieved deep on-target depth of coverage (DoC) at average > 100× and > 98% uniformity using one MinION flowcell. For accurate ONT variant calling, the generated reads sufficiently covered 98.9% of pathogenic positions listed in ClinVar, with 98.96% having at least 30× DoC. ECNano obtained an average read length of 1000 bp. The long reads of ECNano also covered the adjacent splice sites well, with 98.5% of positions having ≥ 30× DoC. Clair-ensemble achieved > 99% recall and accuracy for SNV calling. The whole workflow from wet-lab protocol to variant detection was completed within three days. Conclusion We presented ECNano, an out-of-the-box workflow comprising (1) a wet-lab protocol for ONT target enrichment sequencing and (2) a downstream variant detection workflow, Clair-ensemble. The workflow is cost-effective, with a short turnaround time for high accuracy variant calling in 4800 clinically significant genes and regions using a single MinION flowcell. The long-read exon captured data has potential for further development, promoting the application of long-read sequencing in personalized disease treatment and risk prediction.

Boxwood, a low-maintenance landscape plant, has been plagued by diseases in recent years, and fungicide protection is now indispensable for its healthcare. The objective of present study was to determine how fungicide chemistry and repeated application may affect phyllosphere mycobiome. Three fungicides—Daconil (chlorothalonil, contact chemistry), Banner Maxx (propiconazole, systemic chemistry), and Concert II (a combination of both chemistries)—were first applied on April 12 then repeated at 2- and 3-week intervals, product dependent. Shoots from Buxus sempervirens ‘Vardar Valley’ were sampled immediately before, 1, 7 and 14 days after fungicide application on May 26 and August 25. As determined by amplicon sequencing, fungal community composition differed between shoot surface and internal tissue, with the former being dominated by Cladosporium and the latter by Shiraia species. Fungicide applications strongly affected epiphytic fungal community diversity, structure, and many functional groups. Daconil and Concert II suppressed greater numbers of epiphytes than Banner Maxx. Many epiphytic genera became less sensitive to Daconil treatment in August. This study provided the first mycobiome evidence supporting boxwood as a low-maintenance plant and demonstrating fungicide resistance to a multisite chemistry due to repeated applications. It also helped understand boxwood rising health issues associated with increasing fungicide use.

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

We documented 4 cases of severe acute respiratory syndrome coronavirus 2 reinfection by non-variant of concern strains among healthcare workers in Campinas, Brazil. We isolated infectious particles from nasopharyngeal secretions during both infection episodes. Improved and continued protection measures are necessary to mitigate the risk for reinfection among healthcare workers.

Background Leishmaniasis is a parasitic disease caused by obligate intracellular protozoa of the genus Leishmania . This infection is characterized by a wide range of clinical manifestations, with symptoms greatly dependent on the causal parasitic species. Here we present the design and application of a new 70-kDa heat shock protein gene ( hsp70 )-based marker of 771 bp (HSP70-Long). We evaluated its sensitivity, specificity and diagnostic performance employing an amplicon-based MinION™ DNA sequencing assay to identify different Leishmania species in clinical samples from humans and reservoirs with cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). We also conducted a comparative analysis between our novel marker and a previously published HSP70 marker known as HSP70-Short, which spans 330 bp. Methods A dataset of 27 samples from Colombia, Venezuela and the USA was assembled, of which 26 samples were collected from humans, dogs and cats affected by CL and one sample was collected from a dog with VL in the USA (but originally from Greece). DNA was extracted from each sample and underwent conventional PCR amplification utilizing two distinct HSP70 markers: HSP70-Short and HSP70-Long. The subsequent products were then sequenced using the MinION™ sequencing platform. Results The results highlight the distinct characteristics of the newly devised HSP70-Long primer, showcasing the notable specificity of this primer, although its sensitivity is lower than that of the HSP70-Short marker. Notably, both markers demonstrated strong discriminatory capabilities, not only in distinguishing between different species within the Leishmania genus but also in identifying instances of coinfection. Conclusions This study underscores the outstanding specificity and effectiveness of HSP70-based MinION™ sequencing, in successfully discriminating between diverse Leishmania species and identifying coinfection events within samples sourced from leishmaniasis cases. Graphical abstract

The dissemination of antibiotic resistance (AR) through various environments and the role of AR hotspots in public health crises are gaining increasing attention. Aquatic biofilms are speculated to play a significant role in AR spread due to their collection of diverse microorganisms and facilitation of horizontal gene transfer (HGT). However, few studies have characterized the AR genes (resistome) present in natural river biofilms. The goal of this study was to use MinION long-read sequencing to analyze the microbiome, resistome, and mobile genetic elements (MGEs) in periphyton (epilithic biofilms) ( n = 56) from a multiuse watershed in Ohio, to elucidate the role of periphyton in clinically relevant AR. Key members of the periphyton microbiome included Flavobacterium and Aeromonas . Overall, periphyton microbial communities shifted with season and location. Specifically, species of Porphyrobacter and Cyanobacteria were more abundant in biofilms during the summer season. Potentially pathogenic bacteria, including the family Enterobacteriaceae , the fish pathogen Pseudomonas koreensis , and the human pathogen Shigella flexneri , were more abundant in sites downstream of the large city, Columbus, OH, than upstream. The periphyton resistome carried diverse AR genes for a variety of classes, but had minimal clinical relevance. Escherichia , Escherichia coli , and Muvirus were common hosts of AR genes (ARGs) and MGEs. Pseudomonas and Cyanobacteria were frequently MGE hosts, but not AR genes, indicating the potentially important role of these taxa in HGT within and around biofilms. While the sequencing depth in this study was relatively shallow, these findings highlight the mobility potential for the transmission of ARGs in river biofilms.