Oxford Nanopore Technologies MinION Research Articles

Rapid Whole Genome Characterization of High-Risk Pathogens Using Long-Read Sequencing to Identify Potential Healthcare Transmission.

Routine use of whole genome sequencing (WGS) has been shown to help identify transmission of pathogens causing healthcare-associated infections (HAIs). However, the current gold standard of short-read, Illumina-based WGS is labor and time-intensive. In light of recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing, we sought to establish a low resource utilization approach capable of providing accurate WGS-based comparisons of HAI pathogens within a time frame allowing for infection prevention and control (IPC) interventions. WGS was prospectively performed on antimicrobial-resistant pathogens at increased risk of potential healthcare transmission using the ONT MinION sequencer with R10.4.1 flow cells and Dorado basecalling algorithm. Potential transmission was assessed via Ridom SeqSphere+ for core genome multilocus sequence typing and MINTyper for reference-based core genome single nucleotide polymorphisms using previously published cut-off values. The accuracy of our ONT pipeline was determined relative to Illumina-based WGS data generated from the same genomic DNA sample. Over a six-month period, 242 bacterial isolates from 216 patients were sequenced by a single operator. Compared to the Illumina gold-standard data, our ONT pipeline achieved a Q score of 60 for assembled genomes, even with a coverage rate of as low as 40X. The mean time from initiating DNA extraction to complete genetic analysis was 2 days (IQR 2-3.25 days). We identified five potential transmission clusters comprising 21 isolates (8.7% of all sequenced strains). Combining ONT WGS data with epidemiological data, >70% (15/21) of the isolates originated from patients with potential healthcare transmission links. Via a stand-alone ONT pipeline, we detected potentially transmitted HAI pathogens rapidly and accurately, aligning closely with epidemiological data. Our low-resource method has the potential to assist in the efficient detection and deployment of preventative measures against HAI transmission.

Accurate and comprehensive evaluation of O6-methylguanine-DNA methyltransferase promoter methylation by nanopore sequencing.

The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter region is essential in evaluating the prognosis and predicting the drug response in patients with glioblastoma. In this study, we evaluated the utility of using nanopore long-read sequencing as a method for assessing methylation levels throughout the MGMT CpG-island, compared its performance to established techniques and demonstrated its clinical applicability. We analysed 165 samples from CNS tumours, focusing on the MGMT CpG-island using nanopore sequencing. Oxford Nanopore Technologies (ONT) MinION and PromethION flow cells were employed for single sample or barcoded assays, guided by a CRISPR/Cas9 protocol, adaptive sampling or as part of a whole genome sequencing assay. Methylation data obtained through nanopore sequencing were compared to results obtained via pyrosequencing and methylation bead arrays. Hierarchical clustering was applied to nanopore sequencing data for patient stratification. Nanopore sequencing displayed a strong correlation (R2 = 0.91) with pyrosequencing results for the four CpGs of MGMT analysed by both methods. The MGMT-STP27 algorithm's classification was effectively reproduced using nanopore data. Unsupervised hierarchical clustering revealed distinct patterns in methylated and unmethylated samples, providing comparable survival prediction capabilities. Nanopore sequencing yielded high-confidence results in a rapid timeframe, typically within hours of sequencing, and extended the analysis to all 98 CpGs of the MGMT CpG-island. This study presents nanopore sequencing as a valid and efficient method for determining MGMT promotor methylation status. It offers a comprehensive view of the MGMT promoter methylation landscape, which enables the identification of potentially clinically relevant subgroups of patients. Further exploration of the clinical implications of patient stratification using nanopore sequencing of MGMT is warranted.

Early identification of a ward-based outbreak of Clostridioides difficile using prospective multilocus sequence type-based Oxford Nanopore genomic surveillance.

To describe an outbreak of sequence type (ST)2 Clostridioides difficile infection (CDI) detected by a recently implemented multilocus sequence type (MLST)-based prospective genomic surveillance system using Oxford Nanopore Technologies (ONT) sequencing. Hemato-oncology ward of a public tertiary referral centre. From February 2022, we began prospectively sequencing all C. difficile isolated from inpatients at our institution on the ONT MinION device, with the output being an MLST. Bed-movement data are used to construct real-time ST-specific incidence charts based on ward exposures over the preceding three months. Between February and October 2022, 76 of 118 (64.4%) CDI cases were successfully sequenced. There was wide ST variation across cases and the hospital, with only four different STs being seen in >4 patients. A clear predominance of ST2 CDI cases emerged among patients with exposure to our hemato-oncology ward between May and October 2022, which totalled ten patients. There was no detectable rise in overall CDI incidence for the ward or hospital due to the outbreak. Following a change in cleaning product to an accelerated hydrogen peroxide wipe and several other interventions, no further outbreak-associated ST2 cases were detected. A retrospective phylogenetic analysis using original sequence data showed clustering of the suspected outbreak cases, with the exception of two cases that were retrospectively excluded from the outbreak. Prospective genomic surveillance of C. difficile using ONT sequencing permitted the identification of an outbreak of ST2 CDI that would have otherwise gone undetected.

Whole genome sequencing analysis on antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia.

The emergence and rapid increase in the incidence of multidrug-resistant (MDR) bacteria in pig farms has become a serious concern and reduced the choice of effective antibiotics. This study analyzed the phylogenetics and diversity of antibiotic resistance genes (ARGs) and molecularly identified the source of ARGs in antibiotic-resistant Escherichia coli isolated from pig farms in Banten Province, Indonesia. Forty-four antibiotic-resistant E. coli isolates from fecal samples from 44 pig farms in Banten Province, Indonesia, were used as samples. The samples were categorized into 14 clusters. Sequencing was performed using the Oxford Nanopore Technologies MinION platform, with barcoding before sequencing with Nanopore Rapid sequencing gDNA-barcoding (SQK-RBK110.96) according to manufacturing procedures. ARG detection was conducted using ResFinder, and the plasmid replicon was determined using PlasmidFinder. Three phylogenetic leaves of E. coli were identified in the pig farming cluster in Banten Province. The E. coli isolates exhibited potential resistance to nine classes of antibiotics. Fifty-one ARGs were identified across all isolates, with each cluster carrying a minimum of 10 ARGs. The ant(3'')-Ia and qnrS1 genes were present in all isolates. ARGs in the E. coli pig farming cluster originated mainly from plasmids, accounting for an average of 89.4%. The elevated potential for MDR events, coupled with the dominance of ARGs originating from plasmids, increases the risk of ARG spread among bacterial populations in animals, humans, and the environment.

Genetic Analyses of Rare ESBL ST628 Klebsiella pneumoniae Detected during a Protracted Nosocomial Outbreak in the United Kingdom.

Klebsiella pneumoniae (K. pneumoniae) cultures from a hospital-wide outbreak in the UK, which lasted for over 12 months, were sequenced. We sought to sequence and genetically characterise the outbreak strain. Antibiotic Susceptibility Testing (AST) was performed on 65 K. pneumoniae isolates saved from the outbreak. All isolates were sequenced using the Oxford Nanopore Technologies (ONT) MinION flowcell: 10 isolates, including the isolate with the earliest collection date in 2017, were additionally sequenced on the NovaSeq 6000 platform to build high-accuracy nanopore-illumina assemblies. Among the sequenced strains, 60 were typed as ST628. 96.6% (n = 58/60) ST628 strains harboured a large ~247-kb FIB(K) plasmid carrying up to 11 antimicrobial resistance genes, including the extended-spectrum beta-lactamase (ESBL) gene, blaCTX-M-15. Clonality between the outbreak isolates was confirmed using single nucleotide polymorphism (SNP) typing. The outbreak strains were phylogenetically related to clinical ST628 strains identified in 2012, 6 years prior to the outbreak. A rare ESBL K. pneumoniae K2 ST628 strain harbouring a multi-drug resistant (MDR) plasmid encoding the ESBL gene blaCTX-M-15 was detected across multiple independent wards during the protracted nosocomial outbreak. Surveillance of this strain is recommended to prevent future nosocomial outbreaks.

PHARE: a bioinformatics pipeline for compositional profiling of multiclonal Plasmodium falciparum infections from long-read Nanopore sequencing data.

The emergence of drug-resistant clones of Plasmodium falciparum is a major public health concern, and the ability to detect and track the spread of these clones is crucial for effective malaria control and treatment. However, in endemic settings, malaria infected people often carry multiple P. falciparum clones simultaneously making it likely to miss drug-resistant clones using traditional molecular typing methods. Our goal was to develop a bioinformatics pipeline for compositional profiling in multiclonal P. falciparum samples, sequenced using the Oxford Nanopore Technologies MinION platform. We developed the 'Finding P. falciparum haplotypes with resistance mutations in polyclonal infections' (PHARE) pipeline using existing bioinformatics tools and custom scripts written in python. PHARE was validated on three control datasets containing P. falciparum DNA of four laboratory strains at varying mixing ratios. Additionally, the pipeline was tested on clinical samples from children admitted to a paediatric hospital in the Central African Republic. The PHARE pipeline achieved high recall and accuracy rates in all control datasets. The pipeline can be used on any gene and was tested with amplicons of the P. falciparum drug resistance marker genes pfdhps, pfdhfr and pfK13. The PHARE pipeline helps to provide a more complete picture of drug resistance in the circulating P. falciparum population and can help to guide treatment recommendations. PHARE is freely available under the GNU Lesser General Public License v.3.0 on GitHub: https://github.com/Fippu/PHARE.

Comparing DNA isolation and sequencing strategies for 16S rRNA gene amplicon analysis in biofilm containing environments

Bacteria are primarily responsible for biological water treatment processes in constructed wetland systems. Gravel in constructed wetlands serves as an essential substrate onto which complex bacterial biofilms may successfully grow and evolve. To fully understand the bacterial community in these systems it is crucial to properly isolate biofilms and process DNA from such substrates. This study looked at how best to isolate bacterial biofilms from gravel substrates in terms of bacterial richness. It considered factors including the duration of agitation during extraction, extraction temperature, and enzyme usage. Further, the 16S taxonomy data subsequently produced from Illumina MiSeq reads (using the SILVA 132 ribosomal RNA (rRNA) database on the DADA2 pipeline) were compared with the 16S data produced from Oxford Nanopore Technologies (ONT) MinION reads (using the NCBI 16S database on the EPI2ME pipeline). Finally, performance was tested by comparing the taxonomy data generated from the Illumina MiSeq and ONT MinION reads using the same (SILVA 132) database. We found no significant differences in the effective number of species observed when using different bacterial biofilm detachment techniques. However, enzyme treatment enhanced the total concentration of DNA. In terms of wetland community profiles, relative abundance differences within each sample type were clearer at the genus level. For genus-level taxonomic classification, MinION sequencing with the EPI2ME pipeline (NCBI database) produced bacterial abundance information that was poorly correlated with that from the Illumina MiSeq and DADA2 pipelines (SILVA132 database). When using the same database for each sequencing technology (SILVA132), the correlation between relative abundances at genus-level improved from negligible to moderate. This study provides detailed information of value to researchers working on constructed wetlands regarding efficient biofilm detachment techniques for DNA isolation and 16 s metabarcoding platforms for sequencing and data analysis.

Chromosome-Level Assemblies for the Pine Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen, Fusarium circinatum, is globally regarded as one of the most important threats to commercial pine-based forestry. Although genome sequences of this fungus are available, these remain highly fragmented or structurally ill-defined. Our overall goal was to provide high-quality assemblies for two notable strains of F. circinatum, and to characterize these in terms of coding content, repetitiveness and the position of telomeres and centromeres. For this purpose, we used Oxford Nanopore Technologies MinION long-read sequences, as well as Illumina short sequence reads. By leveraging the genomic synteny inherent to F. circinatum and its close relatives, these sequence reads were assembled to chromosome level, where contiguous sequences mostly spanned from telomere to telomere. Comparative analyses unveiled remarkable variability in the twelfth and smallest chromosome, which is known to be dispensable. It presented a striking length polymorphism, with one strain lacking substantial portions from the chromosome's distal and proximal regions. These regions, characterized by a lower gene density, G+C content and an increased prevalence of repetitive elements, contrast starkly with the syntenic segments of the chromosome, as well as with the core chromosomes. We propose that these unusual regions might have arisen or expanded due to the presence of transposable elements. A comparison of the overall chromosome structure revealed that centromeric elements often underpin intrachromosomal differences between F. circinatum strains, especially at chromosomal breakpoints. This suggests a potential role for centromeres in shaping the chromosomal architecture of F. circinatum and its relatives. The publicly available genome data generated here, together with the detailed metadata provided, represent essential resources for future studies of this important plant pathogen.

High quality genomes produced from single MinION flow cells clarify polyploid and demographic histories of critically endangered Fraxinus (ash) species

With populations of threatened and endangered species declining worldwide, efforts are being made to generate high quality genomic records of these species before they are lost forever. Here, we demonstrate that data from single Oxford Nanopore Technologies (ONT) MinION flow cells can, even in the absence of highly accurate short DNA-read polishing, produce high quality de novo plant genome assemblies adequate for downstream analyses, such as synteny and ploidy evaluations, paleodemographic analyses, and phylogenomics. This study focuses on three North American ash tree species in the genus Fraxinus (Oleaceae) that were recently added to the International Union for Conservation of Nature (IUCN) Red List as critically endangered. Our results support a hexaploidy event at the base of the Oleaceae as well as a subsequent whole genome duplication shared by Syringa, Osmanthus, Olea, and Fraxinus. Finally, we demonstrate the use of ONT long-read sequencing data to reveal patterns in demographic history.

The Oxford Nanopore MinION as a Versatile Technology for the Diagnosis and Characterization of Emerging Plant Viruses.

The emergence of novel viral epidemics that could affect major crops represents a serious threat to global food security. The early and accurate identification of the causative viral agent is the most important step for a rapid and effective response to disease outbreaks. Over the last years, the Oxford Nanopore Technologies (ONT) MinION sequencer has been proposed as an effective diagnostic tool for the early detection and identification of emerging viruses in plants, providing many advantages compared with different high-throughput sequencing (HTS) technologies. Here, we provide a step-by-step protocol that we optimized to obtain the virome of "Lamon bean" plants (Phaseolus vulgaris L.), an agricultural product with Protected Geographical Indication (PGI) in North-East of Italy, which is frequently subjected to multiple infections caused by different RNA viruses. The conversion of viral RNA in ds-cDNA enabled the use of Genomic DNA Ligation Sequencing Kit and Native Barcoding DNA Kit, which have been originally developed for DNA sequencing. This allowed the simultaneous diagnosis of both DNA- and RNA-based pathogens, providing a more versatile alternative to the use of direct RNA and/or direct cDNA sequencing kits.

Mycobiome Analysis of Tall Fescue Grass Under Drought Stress Using the Illumina MiSeq and Oxford Nanopore Technology MinION

The effects that mycobiomes have on physiological traits in turfgrasses are poorly understood. Drought tolerance, an economically and ecologically important trait, can be influenced by symbiotic fungi. In this two-year study, we evaluated the mycobiome associated with tall fescue exposed to prolonged periods of drought stress in a rainout shelter. Twelve plants, comprising six sets of half-sibs (progenies having one parent in common), one exhibiting a drought-tolerant phenotype and the other a drought-susceptible phenotype, were selected for analysis each year. The mycobiomes associated with the shoot, root, and rhizosphere soil were evaluated for each tall fescue half-sib pair using both short-read Illumina MiSeq and long-read Oxford Nanopore Technology (ONT) MinION sequencing pipelines. Both platforms sequenced portions of the fungal nuclear ribosomal RNA genes. The Illumina MiSeq sequenced the internal transcribed spacer region (ITS, 600 bp), while the ONT MinION covered the small subunit, ITS, and partial large subunit (4,600 bp). Both sequencing pipelines revealed that the mycobiomes associated with the roots, shoots, and soil were significantly different. The ONT MinION pipeline identified more diverse fungal lineages and had higher taxonomic resolution than the Illumina pipeline. Our results also indicated that root pathogens may play a more important role than endophytic or mycorrhizal symbionts in tall fescue drought stress tolerance.

Development of a portable on-site applicable metagenomic data generation workflow for enhanced pathogen and antimicrobial resistance surveillance

Rapid, accurate and comprehensive diagnostics are essential for outbreak prevention and pathogen surveillance. Real-time, on-site metagenomics on miniaturized devices, such as Oxford Nanopore Technologies MinION sequencing, could provide a promising approach. However, current sample preparation protocols often require substantial equipment and dedicated laboratories, limiting their use. In this study, we developed a rapid on-site applicable DNA extraction and library preparation approach for nanopore sequencing, using portable devices. The optimized method consists of a portable mechanical lysis approach followed by magnetic bead-based DNA purification and automated sequencing library preparation, and resulted in a throughput comparable to a current optimal, laboratory-based protocol using enzymatic digestion to lyse cells. By using spike-in reference communities, we compared the on-site method with other workflows, and demonstrated reliable taxonomic profiling, despite method-specific biases. We also demonstrated the added value of long-read sequencing by recovering reads containing full-length antimicrobial resistance genes, and attributing them to a host species based on the additional genomic information they contain. Our method may provide a rapid, widely-applicable approach for microbial detection and surveillance in a variety of on-site settings.

PSXIII-20 The Effect of Hemp Varieties and Cannabidiol Concentrations on the in Vitro Ruminal Microbiome

Abstract This study aimed to evaluate the effect of hemp varieties and cannabidiol (CBD) concentrations on in vitro ruminal microbiota diversity and abundance relative do alfalfa (Medicago sativa). Two ruminally cannulated Angus steers were used as inoculum donors for in vitro incubation. Two 160-mL serum bottles containing 200 mg air-dried of either alfalfa, fedora 17 (F17) and felina 32 after late harvest (F32S) or without seeds (F32O) were incubated in 14 mL of buffering media and 4 mL of rumen inoculum. A stock solution of 1mg/mL of CBD was prepared and added in increasing concentrations into each bottle to reach a final concentration of 0.001%, 0.002%, 0.003%, and 0.004% of CBD. After a period of 48 hours, the whole content inside of the serum bottles was used immediately for DNA extraction followed by 16S rRNA sequencing with an Oxford Nanopore technologies MiniON. Amplicon sequence data were bioinformatically processed in QIIME2 version 2022.2. Alpha diversity was measured by the Shannon diversity index. Bray-Curtis’s dissimilarity matrix was constructed to estimate the differences among the samples and CBD levels, and differential abundance was then evaluated using analysis of compositions of microbiomes with bias correction (ANCOM) testing. Statistical significance was established at P < 0.05. All data were visualized in R version 4.1.2 (R Core Team, 2022) using the qiime2R package. Shannon diversity index was the highest for alfalfa (P < 0.05), but no difference was observed among the different CBD inclusion levels. When looking at beta diversity through Bray-Curtis, differences were only observed when comparing the different alfalfa and hemp varieties (P < 0.05), where alfalfa grouped separately from all the hemp samples. The most abundant phyla included Firmicute, Bacteroidetes, Proteobacteria, and Fusobacteria (P < 0.05). At family level, alfalfa samples had greater Aerococcaceae and Enterobacteriaceae than all the hemp treatments, but less Lactobacillales (unclassified), Streptococcaceae, Succinivibrionaceae, and Bacilli (unclassified) than F32S and F17 (P < 0.05). In conclusion, these results suggest that sample type might play a larger role on microbiome than CBD lconcentrations. Further studies are required to determine if greater concentrations of CBD than those examined herein could also lead to a shift on rumen microbes.

Feasibility and Accuracy of Nanopore Transcriptome Sequencing for Pediatric Acute Leukemia Lineage Classification in a Resource-Constrained Setting

Introduction: Gene expression profiling (GEP) has the potential to offer alternative approaches to conventional testing methodologies (flow cytometry, immunohistochemistry, karyotyping, and FISH) for cancer diagnostics. Possible advantages of this approach include unified protocols, automated analyses obviating high-end technical expertise, and perhaps lower training, capital, and skilled human resource requirements in comparison with complex traditional testing. These factors are of particular value in resource-constrained settings, where even leukemia lineage classification is unavailable or inconsistent in many locations, with critical implications for risk stratification and treatment selection. The goals of this study were to (a) ascertain and subsequently improve the accuracy of a lineage classifier in a different context than the pre-testing stage, and (b) determine feasibility of mRNA sequencing for cancer classification in a low-resource setting. The approach utilized PCR-amplified full-length cDNA sequencing using Oxford Nanopore Technologies (ONT) PCR-cDNA barcoding kit (SQK-PCB109) and MinION Mk1b. Methods: One hundred and ten pediatric acute leukemia specimens were collected and sequenced at Indus Hospital & Health Network (IHHN), Karachi, Pakistan and analyzed at the University of North Carolina at Chapel Hill (UNC), USA. Specimens (isolated peripheral blood and / or bone marrow mononuclear cells), frozen in liquid nitrogen or Zymo DNA/RNA shield at -80 oC, were acquired from IHHN's pediatric biorepository. Specimen-associated data included clinical history, CBC, flow cytometry, FISH and karyotyping results from routine clinical testing at the IHHN's ISO 15189-accredited clinical laboratory. The study site did not have prior experience with NGS and did not require external hands-on training for this project. 100 ng of extracted RNA from each of 48 B-ALL, 30 T-ALL, 31 AML, and 1 mixed phenotype acute leukemia specimens was used to prepare libraries. Sequencing data were transferred to UNC and analyzed with an acute leukemia lineage classification algorithm. Results: Following delivery of reagents and equipment, a 6-week protocol optimization and self-training phase utilized open-source protocols and training videos. The next runs to sequence 110 specimens were completed in 4 months by 2 technical staff at the study site. Using a probability threshold for confident calling (>0.8), established during the development phase at UNC, 91 of 110 specimens (82.7%) were called with high confidence. Of the confident calls, 85 were correctly classified (42 B-ALL, 21 T-ALL, and 22 AML), representing an accuracy of 93.4%. The read N50 of confidently classified, concordant specimens ranged from 270 to 745. The total number of reads ranged from 10,000 to >900,000, while the number of aligned reads ranged from 5,000 to 1.5 million. On the other hand, 6 (6.6%) confidently called specimens were discordant. Of these, the mixed phenotype specimen was classified as AML, but mixed phenotype acute leukemia is not included in our training dataset. One misclassified T-ALL specimen had a read count below 10,000 - a threshold demonstrated in the development phase to be optimal for correct classification. Two additional misclassified samples had very low diversity of transcripts express, which is a quality indicator in development. Read counts of these specimens ranged from 22,000 to 311,000, while aligned reads were between 10,000 and 293,000. Further investigation of these discordant calls is ongoing. Low confidence calls (17% of total), with a classification probability between less than 0.8, were either concordant (13) or discordant (6). Further investigation and retesting of these specimens to ascertain reproducibility of low confidence calls has not been performed yet. Conclusions: Taken together, the approach tested - with automated analysis, limited space, training and technical staffing requirements - was feasible at the study site. It provides proof of concept for a low-cost, ONT sequencing based GEP approach for acute leukemia diagnostics whereby testing can be performed in peripheral laboratories while analysis can be centralized, even across continents. Next steps include (a) incorporating these data into a new training model with updated quality control parameters for the next validation run, and (b) developing the genomic subtype classification results.

Draft genome sequence of Janibacter limosus strain RCAM05316 isolated from Shulgan-Tash cave lime mud.

The Shulgan-Tash (Kapova) cave is a unique object for scientific research. In this article, we report the draft genome sequence of Janibacter limosus strain P1(28)-3 (RCAM05316) isolated from cave lime mud, Russia (53° 2' 0″ N, 57° 3' 0″ E). The sequence was obtained using Oxford Nanopore Technologies MinION.

A novel eDNA approach for rare species monitoring: Application of long-read shotgun sequencing to Lynx rufus soil pawprints

Environmental DNA (eDNA) sampling is a relatively new technique that has been employed in biodiversity surveys around the world. Environmental DNA can be an effective, non-invasive method of identifying the presence of target host species. Furthermore, advances in DNA deep sequencing technologies are enabling in-depth information to be recovered from eDNA. Here we report the development of a bobcat (Lynx rufus) species-specific probe-based qPCR assay for eDNA studies, which was validated on wild samples and those from a bobcat housed at the Jacksonville Zoo in Florida. Furthermore, we show that long-read shotgun sequencing of eDNA extracted from pawprint soil samples, using an Oxford Nanopore Technologies MinION, could successfully detect bobcat DNA, including enabling correct species level identification and phylogenetic placement within regionally distanced bobcats. This was all achieved without utilizing any DNA enrichment approaches. The long read shotgun sequencing simultaneously recovered genetic information from microbes known to be part of the bobcat microbiome, indicating that potential health-status-related microbial information can be obtained alongside wildlife eDNA. This study revealed that non-targeted (no metabarcoding or enrichment) long-read shotgun sequencing of eDNA samples can be sufficient for species identification, phylogenetic analyses, and population genetics applications. This paves the way for the rapid deployment of eDNA approaches for any species of interest, without requiring laborious development of targeted approaches, thereby increasing the ease and utility of eDNA research for endangered species conservation and management.

First report of Fig virus B in Ficus carica in New Zealand.

Fig (Ficus carica) has been cultivated since ancient times, and is now grown worldwide, both for its fruit and as an ornamental plant. Several viruses and viroids are associated with Fig mosaic disease (FMD), a disease complex occurring worldwide (Preising et al. 2021). Fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig mild mottle-associated virus (FMMaV), and fig badnavirus 1 (FBV-1) are known to infect fig in New Zealand (Minafra et al. 2012; Veerakone et al. 2015). In December 2020, leaf samples from a fig tree growing on the roadside at St Heliers, Auckland, showing dieback with foliar chlorotic mosaic symptoms, was received for virus testing. Total nucleic acid was extracted from the symptomatic leaves using a KingFisher™ mL Purification System (Thermofisher Scientific, Waltham, MA) with an InviMag Plant DNA Mini Kit (Invitek Molecular GmbH, Germany) and subjected to high-throughput sequencing on an Oxford Nanopore Technologies MinION device using the method described in Liefting et al. 2021. All sequence analysis was performed using Geneious Prime 2021.1.1 (https://www.geneious.com). A total of 355,858 reads that passed quality check were subjected to BLASTn search against the NCBI nt database as described in Liefting et al. 2021. The following viruses produced hits: FMV, FBV-1, FMMaV and a fig closterovirus. The presence of FMV, FBV-1 and FMMaV were confirmed by species specific RT-PCRs. To identify the closterovirus, reads were mapped to closteroviruses reported in fig including the recently identified tentative species fig virus A (FiVA; GenBank accession no MN817232) and fig virus B (FiVB; GenBank accession no. MN817233). Five viral contigs ranging from 939 to 2,340 nucleotides (nt) were obtained from mapping to FiVB. Subsequently, a 6.4 kb sequence (GenBank accession no. OQ968551) from the 3' region of the NZ isolate was amplified by overlapping RT-PCR using primers designed from the contig sequences. The sequence shared 79.5% nucleotide (nt) identity with FiVB The original sample and a further 25 symptomatic and 10 asymptomatic fig samples, collected from the Auckland area between 2016 and 2021, were tested using FiVB specific RT-PCR and Sanger sequencing using primers FiVB-F1 (5'-GAGGGAGAGATGTAGATGC-3') and FiVB-R2 (5'-TGTCGTCGATATCGTTGTGT-3'), designed to amplify a 725 nt fragment in the 70 kDa heat shock protein (HSP70) ORF. Products of the expected size were amplified from the original sample and three symptomatic samples and their sequences found to be identical. BLAST searches showed that the sequence (GenBank accession no. ON553403) shared 82.7% nt and 87.3% amino acid (aa) identity to an isolate of FiVB (GenBank accession no MN817233). These additional positive samples were collected from a small home nursery where the plants were propagated from cuttings and have been distributed locally, suggesting the virus is very likely to have a limited spread throughout the Auckland area. All three FiVB infected samples were also positive for FMV. However, the association of FiVB with FMD symptoms is unknown. FiVB was first identified from a latex sample exuded from a fig tree collected from Japan (Park et al. 2021) and is the only report of FiVB in the world to date. Although an identical sequence from Argentina, named fig closterovirus 1, was submitted to GenBank, the origin of this isolate is not known. To our knowledge, this is the first report of FiVB in New Zealand.

Comparison of nine extraction methods for bacterial identification using the ONT MinION sequencer.

The Anthrax mailings bioterrorism attack in 2001 revealed the need for universal and rapid microbial forensic analyses on unknown biological evidence. However, the gold standard for bacterial identification includes culturing isolates, which is laborious. Molecular approaches for bacterial identification revolve around 16S ribosomal gene sequencing using Sanger or next generation sequencing (NGS) platforms, but these techniques are laboratory-based and can also be time-consuming. The Oxford Nanopore Technologies (ONT) MinION sequencer can generate long read lengths that span the entire bacterial 16S rRNA gene and accurately identify the species level. This platform can be used in the field, allowing on-site evidence analysis. However, it requires higher quantities of pure DNA compared to other sequencing platforms; thus, the extraction method for bacterial DNA is critical for downstream analysis, which to date are tailored toward a priori knowledge of the species' taxonomic grouping. During an attack, the investigative team may not know what species they are handling; therefore, identifying an extraction method that can handle all bacterial groups and generate clean DNA for the MinION is useful for microbial forensic analysis. The purpose of this study was to identify a "universal" extraction method that can be coupled with ONT MinION sequencing for use in forensic situations for rapid identification. It also evaluated the cloud-based data analysis software provided by ONT, EPI2ME. No "universal" extraction method was identified as optimal for downstream MinION sequencing. However, the DNeasy PowerSoil Kit and Noda et al. Chelex-100 method gave comparable sequencing results and could be used as rapid extraction techniques. This study showed that the ONT 16S Barcoding Kit 1-24 coupled with the 16S FASTQ workflow might not be the best for use in forensic situations where species-level identification needs to be obtained, as most alignments were approximately 89% accurate. In all seven test organisms and nine extraction methods, accurate species identification was onlyobtained in 63% of the cases.

Complete genome assembly of Hawai'i environmental nontuberculous mycobacteria reveals unexpected co-isolation with methylobacteria.

Nontuberculous mycobacteria (NTM) are ubiquitous environmental opportunistic pathogens that can cause chronic lung disease. Within the United States, Hawai'i has the highest incidence of NTM lung disease, though the precise reasons are yet to be fully elucidated. One possibility is the high prevalence of NTM in the Hawai'i environment acting as a potential reservoir for opportunistic NTM infections. Through our previous initiatives to collect and characterize NTM in Hawai'i, community scientists of Hawai'i have collected thousands of environmental samples for sequencing. Here, these community scientists were invited for the first time into a high school lab in O'ahu for a genomic sequencing workshop, where participants sequenced four of the collected isolate genomic samples using the Oxford Nanopore Technologies MinION sequencer. Participants generated high quality long read data that when combined with short read Illumina data yielded complete bacterial genomic assemblies suitable for in-depth analysis. The gene annotation analysis identified a suite of genes that might help NTM thrive in the Hawai'i environment. Further, we found evidence of co-occurring methylobacteria, revealed from the sequencing data, suggesting that in some cases methylobacteria and NTM may coexist in the same niche, challenging previously accepted paradigms. The sequencing efforts presented here generated novel insights regarding the potential survival strategies and microbial interactions of NTM in the geographic hot spot of Hawai'i. We highlight the contributions of community scientists and present an activity that can be reimplemented as a workshop or classroom activity by other research groups to engage their local communities.

Characterisation of the Paenarthrobacter nicotinovorans ATCC 49919 genome and identification of several strains harbouring a highly syntenic nic-genes cluster

BackgroundPaenarthrobacter nicotinovorans ATCC 49919 uses the pyridine-pathway to degrade nicotine and could provide a renewable source of precursors from nicotine-containing waste as well as a model for studying the molecular evolution of catabolic pathways and their spread by horizontal gene transfer via soil bacterial plasmids.ResultsIn the present study, the strain was sequenced using the Illumina NovaSeq 6000 and Oxford Nanopore Technology (ONT) MinION platforms. Following hybrid assembly with Unicycler, the complete genome sequence of the strain was obtained and used as reference for whole-genome-based phylogeny analyses. A total of 64 related genomes were analysed; five Arthrobacter strains showed both digital DNA-DNA hybridization and average nucleotide identity values over the species threshold when compared to P. nicotinovorans ATCC 49919. Five plasmids and two contigs belonging to Arthrobacter and Paenarthrobacter strains were shown to be virtually identical with the pAO1 plasmid of Paenarthrobacter nicotinovorans ATCC 49919. Moreover, a highly syntenic nic-genes cluster was identified on five plasmids, one contig and three chromosomes. The nic-genes cluster contains two major locally collinear blocks that appear to form a putative catabolic transposon. Although the origins of the nic-genes cluster and the putative transposon still elude us, we hypothesise here that the ATCC 49919 strain most probably evolved from Paenarthrobacter sp. YJN-D or a very closely related strain by acquiring the pAO1 megaplasmid and the nicotine degradation pathway.ConclusionsThe data presented here offers another snapshot into the evolution of plasmids harboured by Arthrobacter and Paenarthrobacter species and their role in the spread of metabolic traits by horizontal gene transfer among related soil bacteria.