Metabarcoding using nanopore sequencing enables identification of diverse and zoonotic vector-borne pathogens from neglected regions: A case study investigating dogs from Bhutan.

The diversity and prevalence of canine vector-borne pathogens (VBPs) in Bhutan have to date remained unexplored, whilst recent epidemiological surveys in other South Asian nations have found diseases caused by VBPs to be rife in local dog populations. Importantly, many of such VBPs can infect people as well, with a building body of evidence identifying potentially zoonotic rickettsial organisms infecting humans in Bhutan. Given the lack of data on canine pathogens in Bhutan we employed a suite of deep-sequencing metabarcoding methods using Oxford Nanopore Technologies' MinION™ device to holistically characterise the bacterial, apicomplexan and filarial worm blood-borne pathogens of dogs in the country's south. Of the 95 stray, owned and community dogs sampled 78% (95% CI = 69%-85%) were infected with at least one VBP. Pathogen species detected were highly diverse including the bacteria Mycoplasma haemocanis in 16% (95% CI: 10-24%), Ehrlichia canis in 4% (95% CI: 2-10%), Anaplasma platys in 2% (95% CI: 0.5-7%) of dogs as well as the zoonotic species Bartonella clarridgeiae in 1% (95% CI: 0.1-6%), a potentially novel Bartonella spp. and an Ehrlichia chaffeensis-like bacterium, both in 1% (95% CI: 0.1-6%) of dogs. The apicomplexan haemoparasites Hepatozoon canis in 62% (95% CI: 52-71%), Babesia gibsoni in 45% (95% CI: 36-55%) and Babesia vogeli in 3% (95% CI: 1-9%) of dogs were also detected. Finally, 5% (95% CI: 2-12%) of dogs were found to be infected with the filarioid Acanthocheilonema reconditum and 1% (95% CI: 0.1-6%) with zoonotic Dirofilaria sp. hongkongensis. One canine was found positive to the filarioid Setaria tundra, a species normally found infecting cervids. The elucidated diversity of VBP communities highlights the strength of assumption-free diagnostics, such as metabarcoding, in detecting rare, novel, and unexpected pathogens. This approach to identifying pathogen diversity is of critical importance when investigating regions and populations that have thus far been neglected, with the findings aiding the development of future One Health informed strategies for disease control.

High-quality genome assembly and annotation of five bacteria isolated from the Abu Dhabi sabkha-shore region.

OBJECTIVES: Sabkhas represent polyextreme environments characterized by elevated salinity levels, intense ultraviolet (UV) radiation exposure, and extreme temperature fluctuations. In this study, we present the complete genomes of five bacterial isolates isolated from the sabkha-shore region and investigate their genomic organization and gene annotations. A better understanding of the bacterial genomic organization and genetic adaptations of these bacteria holds promise for engineering microbes with tailored functionalities for diverse industrial and agricultural applications, including bioremediation and promotion of plant growth under salinity stress conditions. DATA DESCRIPTION: We present a comprehensive genome sequencing and annotation of five bacteria (kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11) obtained from the shores of the Abu Dhabi Sabkha region. Initial bacterial identification was conducted through 16 S rDNA amplification and sequencing. Employing a hybrid genome assembly technique combining Illumina short reads (NovaSeq 6000) and Oxford Nanopore long reads (MinION), we obtained complete annotated high-quality gap-free genome sequences. The genome sizes of the kcgeb_sa, kcgeb_sc, kcgeb_sd, kcgeb_S4, and kcgeb_S11 isolates were determined to be 2.4 Mb, 4.1 Mb, 2.9 Mb, 5.05 Mb, and 4.1 Mb, respectively. Our analysis conclusively assigned the bacterial isolates as Staphylococcus capitis (kcgeb_sa), Bacillus spizizenii (kcgeb_sc and kcgeb_S11), Pelagerythrobacter marensis (kcgeb_sd), and Priestia aryabhattai (kcgeb_S4).

Nanopore sequencing of forensic short tandem repeats using QNome of Qitan Technology.

Devices of nanopore sequencing can be highly portable and of low cost. Thus, nanopore sequencing is promising in in-field forensic applications. Previous investigations have demonstrated that nanopore sequencing is feasible for genotyping forensic short tandem repeats (STRs) by using sequencers of Oxford Nanopore Technologies. Recently, Qitan Technology launched a new portable nanopore sequencer and became the second supplier in the world. Here, for the first time, we assess the QNome (QNome-3841) for its accuracy in nanopore sequencing of STRs and compare with MinION (MinION Mk1B). We profile 54 STRs of 21 unrelated individuals and 2800M standard DNA. The overall accuracy for diploid STRs and haploid STRs were 53.5% (378 of 706) and 82.7% (134 of 162), respectively, by using QNome. The accuracies were remarkably lower than those of MinION (diploid STRs, 84.5%; haploid, 90.7%), with a similar amount of sequencing data and identical bioinformatics analysis. Although it was not reliable for diploid STRs typing by using QNome, the haploid STRs were consistently correctly typed. The majority of errors (58.8%) in QNome-based STR typing were one-repeat deviations of repeat units in the error from true allele, related with homopolymers in repeats of STRs.

Rapid genotyping of Toxoplasma gondii isolates via Nanopore-based multi-locus sequencing.

Toxoplasma gondii is an obligate intracellular parasite associated with severe disease, especially in the immunosuppressed. It is also a cause of congenital malformation and abortion in both animals and humans and is considered one of the most important foodborne pathogens worldwide with different strains showing variable distribution and differing pathogenicity. Thus, strain-level differentiation of T. gondii isolates is an essential asset in the understanding of parasite's diversity, geographical distribution, epidemiology and health risk. Here, we designed and implemented an Oxford Nanopore MinION protocol to analyse genomic sequence variation including single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (InDel's) of four different genomic loci, part of protein coding genes SAG2, SAG3, ROP17 and ROP21. This method provided results with the sequencing depth necessary for accurate differentiation of T. gondii strains and represents a rapid approach compared to conventional techniques which we further validated against environmental samples isolated from wild wood mice. In summary, multi-locus sequence typing (MLST) of both highly conserved and more polymorphic areas of the genome, provided robust data for strain classification in a platform ready for further adaption for other strains and pathogens.

Sterile sentinels and MinION sequencing capture active soil microbial communities that differentiate crop rotations.

BACKGROUND: Soil microbial communities are difficult to measure and critical to soil processes. The bulk soil microbiome is highly diverse and spatially heterogeneous, which can make it difficult to detect and monitor the responses of microbial communities to differences or changes in management, such as different crop rotations in agricultural research. Sampling a subset of actively growing microbes should promote monitoring how soil microbial communities respond to management by reducing the variation contributed by high microbial spatial and temporal heterogeneity and less active microbes. We tested an in-growth bag method using sterilized soil in root-excluding mesh, "sterile sentinels," for the capacity to differentiate between crop rotations. We assessed the utility of different incubation times and compared colonized sentinels to concurrently sampled bulk soils for the statistical power to differentiate microbial community composition in low and high diversity crop rotations. We paired this method with Oxford Nanopore MinION sequencing to assess sterile sentinels as a standardized, fast turn-around monitoring method. RESULTS: Compared to bulk soil, sentinels provided greater statistical power to distinguish between crop rotations for bacterial communities and equivalent power for fungal communities. The incubation time did not affect the statistical power to detect treatment differences in community composition, although longer incubation time increased total biomass. Bulk and sentinel soil samples contained shared and unique microbial taxa that were differentially abundant between crop rotations. CONCLUSIONS: Overall, compared to bulk soils, the sentinels captured taxa with copiotrophic or ruderal traits, and plant-associated taxa. The sentinels show promise as a sensitive, scalable method to monitor soil microbial communities and provide information complementary to traditional soil sampling.

First Report of Melon Severe Mosaic Orthotospovirus infecting cucurbits in the United States.

Cucurbits (family Cucurbitaceae) includes globally important fruit and vegetable crops. Virus diseases pose a serious threat to cucurbits, limiting crop quality and yield (Regina et al. 2021). In fall 2023, leaf and fruit samples from two squash plants with chlorotic mosaic symptoms and fruit distortion from Monroe and Pope counties in Arkansas were received for diagnosis at the University of Arkansas Division of Agriculture Plant Clinic. Based on symptoms, samples were assessed for melon severe mosaic orthotospovirus (MeSMV) using the ImmunoStrip® developed for detection of the virus (Agdia® Inc., Elkhart, Indiana). The presence of MeSMV was also confirmed by RT-PCR using the Agdia Tospovirus group PCR primers. An amplicon was sequenced and showed 91% sequence identity to the MESMV type isolate (NC_033834, VE440-A). To further verify the results, nucleic acids from a squash sample from Pope County were extracted as described by Poudel et al. (2013), DNase treated, and sequenced on an Oxford Nanopore MinION as described by Liefting et al. (2021). A total of 25,914 raw reads were analyzed using VirFind (Ho and Tzanetakis 2014), which identified 112 reads mapping to the three segments of MeSMV. Primers for all three RNAs were developed and amplified 638, 650, and 1153 nt of the S, M, and L segments of the virus respectively. The amplicons were sequenced bidirectionally and show 89-93% identity to the type isolate from Mexico (GenBank accessions PP301332-4). MeSMV has only been identified in Mexico and can cause significant losses to honeydew melon, zucchini, and cucumber (Ciuffo et al. 2009). Thus, this is the first report of MeSMV outside Mexico. Given the severity of the symptoms observed in cucurbit crops, the virus poses a potential threat to the cucurbit industry in the United States. Growers should be aware of this virus and take the necessary precautions to prevent its spread in the field.

CRISPR/Cas9 gene targeting plus nanopore DNA sequencing with the plasmid pBR322 in the classroom.

Both nanopore-based DNA sequencing and CRISPR/Cas-based gene editing represent groundbreaking innovations in molecular biology and genomics, offering unprecedented insights into and tools for working with genetic information. For students, reading, editing, and even writing DNA will be part of their everyday life. We have developed a laboratory procedure that includes (i) the biosynthesis of a guide RNA for, (ii) targeting Cas9 to specifically linearize the pBR322 plasmid, and (iii) the identification of the cutting site through nanopore DNA sequencing. The protocol is intentionally kept simple and requires neither living organisms nor biosafety laboratories. We divided the experimental procedures into separate activities to facilitate customization. Assuming access to a well-equipped molecular biology laboratory, an initial investment of approximately $2,700 is necessary. The material costs for each experiment group amount to around $130. Furthermore, we have developed a freely accessible website (https://dnalesen.hs-mittweida.de) for sequence read analysis and visualization, lowering the required computational skills to a minimum. For those with strong computational skills, we provide instructions for terminal-based data processing. With the presented activities, we aim to provide a hands-on experiment that engages students in modern molecular genetics and motivates them to discuss potential implications. The complete experiment can be accomplished within half a day and has been successfully implemented by us at high schools, in teacher training, and at universities. Our tip is to combine CRISPR/Cas gene targeting with nanopore-based DNA sequencing. As a tool, we provide a website that facilitates sequence data analysis and visualization.

A full-length 18S ribosomal DNA metabarcoding approach for determining protist community diversity using Nanopore sequencing.

Protist diversity studies are frequently conducted using DNA metabarcoding methods. Currently, most studies have utilized short read sequences to assess protist diversity. One limitation of using short read sequences is the low resolution of the markers. For better taxonomic resolution longer sequences of the 18S rDNA are required because the full-length has both conserved and hypervariable regions. In this study, a new primer pair combination was used to amplify the full-length 18S rDNA and its efficacy was validated with a test community and then validated with field samples. Full-length sequences obtained with the Nanopore MinION for protist diversity from field samples were compared with Illumina MiSeq V4 and V8-V9 short reads. Sequences generated from the high-throughput sequencers are Amplicon Sequence Variants (ASVs). Metabarcoding results show high congruency among the long reads and short reads in taxonomic annotation at the major taxonomic group level; however, not all taxa could be successfully detected from sequences. Based on the criteria of ≥95% similarity and ≥1000 bp query length, 298 genera were identified by all markers in the field samples, 250 (84%) were detected by 18S, while only 226 (76%) by V4 and 213 (71%) by V8-V9. Of the total 85 dinoflagellate genera observed, 19 genera were not defined by 18S dinoflagellate ASVs compared to only three among the total 52 diatom genera. The discrepancy in this resolution is due to the lack of taxonomically available 18S reference sequences in particular for dinoflagellates. Overall, this preliminary investigation demonstrates that application of the full-length 18S rDNA approach can be successful in field studies.

First transcriptome sequencing, assembly, and annotation dataset for the freshwater angelfish, pterophyllum scalare.

Cichlids are relevant to biological research for their craniofacial variations that are analogous to human structure and associated congenital anomalies. However, only a limited number of cichlids have genetic information available. Investigating cichlids and adding to the body of knowledge about them may provide better insights into studying developmental biology and craniofacial structure. The angelfish, Pterophyllum scalare, is one cichlid for which we lack genetic information including a draft transcriptome assembly. This work is the first to provide a draft transcriptome and annotation using long-read Nanopore sequencing for P. scalare. Total RNA was extracted from angelfish tissue, and a cDNA-PCR library was prepared. Sequencing was performed on a singular R.9.4.1 MinION flow cell for 84 h. Various bioinformatic tools were then employed to assemble the sequencing reads into a transcriptome. The transcriptome was then annotated against various databases. 23 million sequencing reads were collected totalling 21.9 Gb. The N50 sequencing read length was 1255 bp and the mean read length was 938. The data had an initial mean Phred score of 10.04. After assembly, the final transcriptome consists of 98,125 transcripts with a mean length of 1552 and N50 length of 2277. The transcriptome has a completeness of 80.5% as assessed by BUSCO. Functional annotation revealed pathways related to signal transduction, carbohydrate metabolism, and transcription are the most annotated in the transcriptome.

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

DNA barcodes are useful in biodiversity research, but sequencing barcodes with dye termination methods ("Sanger sequencing") has been so time-consuming and expensive that DNA barcodes are not as widely used as they should be. Fortunately, MinION sequencers from Oxford Nanopore Technologies have recently emerged as a cost-effective and efficient alternative for barcoding. MinION barcodes are now suitable for large-scale species discovery and enable specimen identification when the target species are represented in barcode databases. With a MinION, it is possible to obtain 10,000 barcodes from a single flow cell at a cost of less than 0.10 USD per specimen. Additionally, a Flongle flow cell can be used for small projects requiring up to 300 barcodes (0.50 USD per specimen). We here describe a cost-effective laboratory workflow for obtaining tagged amplicons, preparing ONT libraries, sequencing amplicon pools, and analyzing the MinION reads with the software ONTbarcoder. This workflow has been shown to yield highly accurate barcodes that are 99.99% identical to Sanger barcodes. Overall, we propose that the use of MinION for DNA barcoding is an attractive option for all researchers in need of a cost-effective and efficient solution for large-scale species discovery and specimen identification.

Bioaugmentation of anaerobic digesters with the enriched lignin-degrading microbial consortia through a metagenomic approach.

The recalcitrance of lignin impedes the efficient utilization of lignocellulosic biomass, hindering the efficient production of biogas and value-added materials. Despite the emergence of anaerobic digestion as a superior alternative to the aerobic method for lignin processing, achieving its feasibility requires thorough characterization of lignin-degrading anaerobic microorganisms, assessment of their biomethane production potential, and a comprehensive understanding of the degradation pathway. This study aimed to address the aforementioned necessities by bioaugmenting seed sludge with three distinct enriched lignin-degrading microbial consortia at both 25 °C and 37 °C. Enhanced biomethane yields was detected in the bioaugmented digesters, while the highest production was observed as 188 mLN CH4 gVS-1 in digesters operated at 37 °C. Moreover, methane yield showed a significant improvement in the samples at 37 °C ranging from 110% to 141% compared to the control, demonstrating the efficiency of the enriched lignin-degrading microbial community. Temperature and substrate were identified as key factors influencing microbial community dynamics. The observation that microbial communities tended to revert to the initial state after lignin depletion, indicating the stability of the overall microbiota composition in the digesters, is a promising finding for large-scale studies. Noteworthy candidates for lignin degradation, including Sporosarcina psychrophila, Comamonas aquatica, Shewanella baltica, Pseudomonas sp. C27, and Brevefilum fermentans were identified in the bioaugmented samples. PICRUSt2 predictions suggest that the pathway and specific proteins involved in anaerobic lignin degradation might share similarities with those engaged in the degradation of aromatic compounds.

Multiplex PCR method for MinION sequencing of Bagaza virus isolated from wild caught mosquitoes in South Africa.

Bagaza virus (BAGV) is a mosquito-borne orthoflavivirus known to occur in regions of southern Europe, Africa, India and the Middle East. The virus has been associated with neurological disease and fatalities in various wild bird species. Association with human disease is not confirmed although limited serological evidence has suggested human infection. Surveillance programs for screening mosquitoes for evidence of arbovirus infection play an important role in providing information regarding the circulation and spread of viruses in specific regions. BAGV was detected in a mosquito pool during surveillance of mosquitoes collected in central South Africa between November 2019 and March 2023. Homogenized mosquito pools were screened for flaviviral RNA using conventional RT-PCR and virus isolation was attempted on positive samples. BAGV was detected and subsequently isolated using cell culture. A multiplex tiling PCR method for targeted enrichment using a PCR based or amplicon sequencing approach of the complete genome of BAGV was developed and optimized. Primers were designed using alignment of complete genome sequence data retrieved from GenBank to identify suitable primer sites that would generate overlapping fragments spanning the complete genome. Six forward primers and eight reverse primers were identified that target the complete genome and amplified nine overlapping fragments, that ranged in length from 1954 to 2039 with an overlap ranging from 71 to 711 base pairs. The design strategy included multiple forward and reverse primer pairs for the 5' and 3' ends. Phylogenetic analysis with other isolates was performed and BAGV isolate VBD 74/23/3 was shown to share high similarity with previous BAGV isolates from all regions, with genetic distance ranging from 0.026 to 0.083. VBD 74/23/3 was most closely related to previous isolates from southern Africa, ZRU96/16/2 isolated from a post-mortem sample from a pheasant in 2016 and MP-314-NA-2018 isolated from mosquitoes in northwestern Namibia with genetic distance 0.0085 and 0.016 respectively. Currently there is limited complete genome sequence data available for many of the arboviruses circulating in Africa. The multiplex tiling method provided a simple and cost-effective method for obtaining complete genome sequence. This method can be readily applied to other viruses using sequence data from publicly available databases and would have important application facilitating genomic surveillance of arboviruses in low resource countries.

Downregulation of Ribosomal Protein Genes Is Revealed in a Model of Rat Hippocampal Neuronal Culture Activation with GABA(A)R/GlyRa2 Antagonist Picrotoxin.

Long-read transcriptome sequencing provides us with a convenient tool for the thorough study of biological processes such as neuronal plasticity. Here, we aimed to perform transcriptional profiling of rat hippocampal primary neuron cultures after stimulation with picrotoxin (PTX) to further understand molecular mechanisms of neuronal activation. To overcome the limitations of short-read RNA-Seq approaches, we performed an Oxford Nanopore Technologies MinION-based long-read sequencing and transcriptome assembly of rat primary hippocampal culture mRNA at three time points after the PTX activation. We used a specific approach to exclude uncapped mRNAs during sample preparation. Overall, we found 23,652 novel transcripts in comparison to reference annotations, out of which ~6000 were entirely novel and mostly transposon-derived loci. Analysis of differentially expressed genes (DEG) showed that 3046 genes were differentially expressed, of which 2037 were upregulated and 1009 were downregulated at 30 min after the PTX application, with only 446 and 13 genes differentially expressed at 1 h and 5 h time points, respectively. Most notably, multiple genes encoding ribosomal proteins, with a high basal expression level, were downregulated after 30 min incubation with PTX; we suggest that this indicates redistribution of transcriptional resources towards activity-induced genes. Novel loci and isoforms observed in this study may help us further understand the functional mRNA repertoire in neuronal plasticity processes. Together with other NGS techniques, differential gene expression analysis of sequencing data obtained using MinION platform might provide a simple method to optimize further study of neuronal plasticity.

Genome Engineering by RNA-Guided Transposition for Anabaena sp. PCC 7120.

In genome engineering, the integration of incoming DNA has been dependent on enzymes produced by dividing cells, which has been a bottleneck toward increasing DNA insertion frequencies and accuracy. Recently, RNA-guided transposition with CRISPR-associated transposase (CAST) was reported as highly effective and specific in Escherichia coli. Here, we developed Golden Gate vectors to test CAST in filamentous cyanobacteria and to show that it is effective in Anabaena sp. strain PCC 7120. The comparatively large plasmids containing CAST and the engineered transposon were successfully transferred into Anabaena via conjugation using either suicide or replicative plasmids. Single guide (sg) RNA encoding the leading but not the reverse complement strand of the target were effective with the protospacer-associated motif (PAM) sequence included in the sgRNA. In four out of six cases analyzed over two distinct target loci, the insertion site was exactly 63 bases after the PAM. CAST on a replicating plasmid was toxic, which could be used to cure the plasmid. In all six cases analyzed, only the transposon cargo defined by the sequence ranging from left and right elements was inserted at the target loci; therefore, RNA-guided transposition resulted from cut and paste. No endogenous transposons were remobilized by exposure to CAST enzymes. This work is foundational for genome editing by RNA-guided transposition in filamentous cyanobacteria, whether in culture or in complex communities.

Etiological agent and clinical characteristics of haemorrhagic fever with renal syndrome in the southern Republic of Korea: a genomic surveillance study.

OBJECTIVES: High incidences of haemorrhagic fever with renal syndrome (HFRS) have been reported in the southern Republic of Korea (ROK). A distinct southern genotype of Orthohantavirus hantanense (HTNV) was identified in Apodemus agrarius chejuensis on Jeju Island. However, its association with HFRS cases in southern ROK remains elusive. We investigated the potential of the southern HTNV genotype as an etiological agent of HFRS. METHODS: Samples from 22 patients with HFRS and 193 small mammals were collected in the southern ROK. The clinical characteristics of patients infected with the southern HTNV genotype were analysed. Amplicon-based MinION sequencing was employed for southern HTNV from patients and rodents, facilitating subsequent analyses involving phylogenetics and genetic reassortment. RESULTS: High-throughput sequencing of HTNV exhibited higher coverage with a cycle of threshold value below 32, acquiring nearly whole-genome sequences from six patients with HFRS and seven A. agrarius samples. The phylogenetic pattern of patient-derived HTNV demonstrated genetic clustering with HTNV from Apodemus species on Jeju Island and the southern Korean peninsula, revealing genetic reassortment in a single clinical sample between the M and S segments. DISCUSSION: These findings imply that the southern HTNV genotype has the potential to induce HFRS in humans. The phylogenetic inference demonstrates the diverse and dynamic characteristics of the southern HTNV tripartite genomes. Therefore, this study highlights the significance of active surveillance and amplicon sequencing for detecting orthohantavirus infections. It also raises awareness and caution for physicians regarding the emergence of a southern HTNV genotype as a cause of HFRS in the ROK.

Rapid detection of high consequence and emerging viral pathogens in pigs.

Introduction: An increasing emergence of novel animal pathogens has been observed over the last decade. Viruses are a major contributor to the increased emergence and therefore, veterinary surveillance and testing procedures are greatly needed to rapidly and accurately detect high-consequence animal diseases such as Foot and Mouth Disease, Highly Pathogenic Avian Influenza, Classical Swine Fever, and African Swine Fever. The major detection methods for such diseases include real-time PCR assays and pathogen-specific antibodies among others. However, due to genetic drift or -shift in virus genomes, failure to detect such pathogens is a risk with devastating consequences. Additionally, the emergence of novel pathogens with no prior knowledge requires non-biased detection methods for discovery. Methods: Utilizing enrichment techniques coupled with Oxford Nanopore Technologies MinION™ sequencing platform, we developed a sample processing and analysis pipeline to identify DNA and RNA viruses and bacterial pathogens from clinical samples. Results and discussion: The sample processing and analysis pipeline developed allows the identification of both DNA and RNA viruses and bacterial pathogens simultaneously from a single tissue sample and provides results in less than 12 h. Preliminary evaluation of this method using surrogate viruses in different matrices and using clinical samples from animals with unknown disease causality, we demonstrate that this method can be used to simultaneously detect pathogens from multiple domains of life simultaneously with high confidence.

Pilot validation of on-field STR typing and human identity testing by MinION nanopore sequencing.

Nanopore sequencing technology has broad application prospects in forensic medicine due to its small size, portability, fast speed, real-time result analysis capabilities, single-molecule sequencing abilities, and simple operation. Here, we demonstrate for the first time that nanopore sequencing platforms can be used to identify individuals in the field. Through scientific and reasonable design, a nanopore MinION MK1B device and other auxiliary devices are integrated into a portable detection box conducive to individual identification at the accident site. Individual identification of 12 samples could be completed within approximately 24 h by jointly detecting 23 short tandem repeat (STR) loci. Through double-blinded experiments, the genotypes of 49 samples were successfully determined, and the accuracy of the STR genotyping was verified by the gold standard. Specifically, the typing success rate for 1150 genotypes was 95.3%, and the accuracy rate was 86.87%. Although this study focused primarily on demonstrating the feasibility of full-process testing, it can be optimistically predicted that further improvements in bioinformatics workflows and nanopore sequencing technology will help enhance the feasibility of Oxford Nanopore Technologies equipment for real-time individual identification at accident sites.

Whole genome sequencing and phylogenetic analysis of SARS-CoV-2 strains isolated during the COVID-19 pandemic in Nigeria.

Objectives: The emergence and spread of SARS-CoV-2 have stimulated ongoing research into the virus transmission dynamics, circulating variants, and potential mutations. This study was conducted to understand the genomic dynamics of the epidemic in Nigeria. Design: Whole genome sequencing was conducted on SARS-CoV-2 samples collected during the first and second outbreaks using the Oxford Nanopore MinION sequencing platform. Phylogenetic analysis was conducted, and genomes were grouped into different pangolin lineages. Results: The study revealed four circulating SARS-CoV-2 variants. The Alpha (B.1.1.7) variant was the most prevalent (32.7%), followed by Beta (B.1 B.1.1, L.3, and B.1.1.318) (30.8%), Eta (B.1.525) (28.9%), and Delta (B.1.617, AY.1, AY.109, and AY.36) (7.7%). Phylogenetic analysis revealed three clusters with four Nextstrain clades (20I, 20B, 21D, and 21J). The Alpha lineages (B.1.1.7) clustered with references from Italy. The Beta lineages (Clade 20B) (B.11, B.11318, and L3) and sub-lineage B.11 were distinct. Sub-lineage B.11318 is clustered with references from the USA, whereas sub-lineage L3 is clustered with references from Russia, the Philippines, Australia, and Japan. The 21D and 21J, belonging to two Pango lineages, Eta (B.1525) and Delta (B.1.617 and AY.109), showed high genetic similarity. Conclusion: The phylogenetic relatedness of the lineages suggests multiple virus introduction, which could be a source of more virulent, locally adapted variants.

The 28S rRNA RT-qPCR assay for host depletion evaluation to enhance avian virus detection in Illumina and Nanopore sequencing.

Abundant host and bacterial sequences can obscure the detection of less prevalent viruses in untargeted next-generation sequencing (NGS). Efficient removal of these non-targeted sequences is vital for accurate viral detection. This study presents a novel 28S ribosomal RNA (rRNA) RT-qPCR assay designed to assess the efficiency of avian rRNA depletion before conducting costly NGS for the detection of avian RNA viruses. The comprehensive evaluation of this 28S-test focuses on substituting DNase I with alternative DNases in our established depletion protocols and finetuning essential parameters for reliable host rRNA depletion. To validate the effectiveness of the 28S-test, we compared its performance with NGS results obtained from both Illumina and Nanopore sequencing platforms. This evaluation utilized swab samples from chickens infected with highly pathogenic avian influenza virus, subjected to established and modified depletion protocols. Both methods significantly reduced host rRNA levels, but using the alternative DNase had superior performance. Additionally, utilizing the 28S-test, we explored cost- and time-effective strategies, such as reduced probe concentrations and other alternative DNase usage, assessed the impact of filtration pre-treatment, and evaluated various experimental parameters to further optimize the depletion protocol. Our findings underscore the value of the 28S-test in optimizing depletion methods for advancing improvements in avian disease research through NGS.

Bulk RNA-seq Assessment of Murine Spleen Using a Portable MinION Sequencing Device.

Comprehensive profiling of the transcriptome reflects the dynamic changes in complex regulator interactions between genes and proteins in the pathological processes of autoimmune disease. To enable real-time adaptive sampling and run rapid sequencing with maximal expected readings of 50 Gb of data with accessibility to clinicians, basic researchers, and even students, a portable and affordable sequencing device, MinION, was employed in our laboratory for both basic and clinical studies. Here, the workflow of bulk RNA-seq in murine spleen using MinION is introduced. The methodology of both laboratory library preparation and the establishment of a bioinformatic pipeline are included in this chapter.