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Influenza A virus poses significant public health challenges due to its high mutation rate and zoonotic potential. Whole-genome sequencing (WGS) is crucial for monitoring and characterizing these viruses. Oxford Nanopore Technologies (ONT) and Illumina next-generation sequencing platforms are commonly used, with ONT being advantageous for its long-read capabilities, portability, and unique ability to access raw data in real-time during sequencing, making it suitable for rapid outbreak responses. This study optimizes the ONT Ligation Sequencing Influenza A Whole Genome protocol by refining RT-PCR kits, primers, and purification methods, and evaluating automation for high-throughput processing. The alternative RT-PCR kits, combined with alternative primers, significantly improved read depth coverage and reduced short, untargeted reads compared to the original ONT protocol. The improvement was particularly evident in the minimum read depth coverage of polymerase segments, which often face challenges with achieving uniform coverage, displaying higher coverage at the 5' and 3' termini, and lower coverage in the central regions. This optimized protocol for targeted influenza A WGS not only enhances sequencing quality and efficiency, but is applicable to all NGS platforms, making it highly valuable for studying influenza adaptation and improving surveillance. Additionally, this protocol can be further refined and adapted for the sequencing of other pathogens, broadening its utility in various pathogen monitoring and response efforts. [ABSTRACT FROM AUTHOR]

Influenza A virus poses significant public health challenges due to its high mutation rate and zoonotic potential. Whole-genome sequencing (WGS) is crucial for monitoring and characterizing these viruses. Oxford Nanopore Technologies (ONT) and Illumina next-generation sequencing platforms are commonly used, with ONT being advantageous for its long-read capabilities, portability, and unique ability to access raw data in real-time during sequencing, making it suitable for rapid outbreak responses. This study optimizes the ONT Ligation Sequencing Influenza A Whole Genome protocol by refining RT-PCR kits, primers, and purification methods, and evaluating automation for high-throughput processing. The alternative RT-PCR kits, combined with alternative primers, significantly improved read depth coverage and reduced short, untargeted reads compared to the original ONT protocol. The improvement was particularly evident in the minimum read depth coverage of polymerase segments, which often face challenges with achieving uniform coverage, displaying higher coverage at the 5’ and 3’ termini, and lower coverage in the central regions. This optimized protocol for targeted influenza A WGS not only enhances sequencing quality and efficiency, but is applicable to all NGS platforms, making it highly valuable for studying influenza adaptation and improving surveillance. Additionally, this protocol can be further refined and adapted for the sequencing of other pathogens, broadening its utility in various pathogen monitoring and response efforts.

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. [ABSTRACT FROM AUTHOR]

Mosquitoes (Family: Culicidae) are dominant vectors of pathogens, and their surveillance has been incorporated into major disease control programs worldwide. However, routine, species-level identification of mosquitoes is often a bottleneck for management, and Next Generation Sequencing (NGS) platforms and DNA metabarcoding can revolutionize this process. MinION nanopore technologies promise on-site sequencing and rapid sample processing rates ideal for time-sensitive biosurveillance. Here, we benchmark the results of DNA metabarcoding on the MinION against the Illumina MiSeq platform, which is known for its higher sequencing accuracy. We used metazoan COI mini-barcode primers to carry out DNA metabarcoding of mosquito bulk samples caught during a real vector survey, then compared the mosquito species profiles recovered on each sequencing platform. We also tested the influence of using different trap lures, storage methods, and pooling different specimen body parts on the number of species recovered. We report that mosquito species-level identifications were highly congruent between MinION and Illumina (93% overlap). We also find that CO2 gas cylinders outperformed biogenic CO2 sources significantly, by two-fold. Notably, we demonstrated the feasibility of detecting zoonotic reservoirs and pathogen signals from mosquito bulk samples. We present the first use of DNA metabarcoding on the MinION for vector surveillance and discuss future applications.

Introduction: Whole Genome Sequencing (WGS) implementation in food safety laboratories is a significant advancement in food pathogen control and outbreak tracking. However, the initial investment for acquiring next-generation sequencing platforms and the need for bioinformatic skills represented an obstacle for the widespread use of WGS. Long-reading technologies, such as the one developed by Oxford Nanopore Technologies, can be easily implemented with a minor initial investment and with simple protocols that can be performed with basic laboratory equipment. Methods: Herein, we report a simple MinION Galaxy-based workflow with analysis parameters that allow its implementation in food safety laboratories with limited computer resources and without previous knowledge in bioinformatics for rapid Salmonella serotyping, virulence, and identification of antimicrobial resistance genes. For that purpose, the single use Flongle flow cells, along with the MinION Mk1B for WGS, and the community-driven web-based analysis platform Galaxy for bioinformatic analysis was used. Three strains belonging to three different serotypes, monophasic S. Typhimurium, S. Grancanaria, and S. Senftenberg, were sequenced. Results: After 24 h of sequencing, enough coverage was achieved in order to perform de novo assembly in all three strains. After evaluating different tools, Flye de novo assemblies with medaka polishing were shown to be optimal for in silico Salmonella spp. serotyping with SISRT tool followed by antimicrobial and virulence gene identification with ABRicate. Discussion: The implementation of the present workflow in food safety laboratories with limited computer resources allows a rapid characterization of Salmonella spp. isolates. [ABSTRACT FROM AUTHOR]

Mycobacterium marinum (M. marinum) is a non-tuberculous mycobacterium (NTM) that can cause infectious diseases in aquatic animals and humans. Culture-based pathogen detection is the gold standard for diagnosing NTM infection. However, this method is time-consuming and has low positivity rates for fastidious organisms. Oxford Nanopore MinION sequencing is an emerging third-generation sequencing technology that can sequence DNA or RNA directly in a culture-independent manner and offers rapid microbial identification. Further benefits include low cost, short turnaround time, long read lengths, and small equipment size. Nanopore sequencing plays a crucial role in assessing drug resistance, clinical identification of microbes, and monitoring infectious diseases. Some reports on Mycobacterium tuberculosis (MTB) using nanopore sequencing have been published, however, there are few reports on NTM, such as M. marinum. Here, we report the use of nanopore sequencing for the diagnosis of M. marinum. [ABSTRACT FROM AUTHOR]

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.

IntroductionWhole Genome Sequencing (WGS) implementation in food safety laboratories is a significant advancement in food pathogen control and outbreak tracking. However, the initial investment for acquiring next-generation sequencing platforms and the need for bioinformatic skills represented an obstacle for the widespread use of WGS. Long-reading technologies, such as the one developed by Oxford Nanopore Technologies, can be easily implemented with a minor initial investment and with simple protocols that can be performed with basic laboratory equipment.MethodsHerein, we report a simple MinION Galaxy-based workflow with analysis parameters that allow its implementation in food safety laboratories with limited computer resources and without previous knowledge in bioinformatics for rapid Salmonella serotyping, virulence, and identification of antimicrobial resistance genes. For that purpose, the single use Flongle flow cells, along with the MinION Mk1B for WGS, and the community-driven web-based analysis platform Galaxy for bioinformatic analysis was used. Three strains belonging to three different serotypes, monophasic S. Typhimurium, S. Grancanaria, and S. Senftenberg, were sequenced.ResultsAfter 24 h of sequencing, enough coverage was achieved in order to perform de novo assembly in all three strains. After evaluating different tools, Flye de novo assemblies with medaka polishing were shown to be optimal for in silico Salmonella spp. serotyping with SISRT tool followed by antimicrobial and virulence gene identification with ABRicate.DiscussionThe implementation of the present workflow in food safety laboratories with limited computer resources allows a rapid characterization of Salmonella spp. isolates.

Primary mitochondrial diseases are progressive genetic disorders affecting multiple organs and characterized by mitochondrial dysfunction. These disorders can be caused by mutations in nuclear genes coding proteins with mitochondrial localization or by genetic defects in the mitochondrial genome (mtDNA). The latter include point pathogenic variants and large-scale deletions/rearrangements. MtDNA molecules with the wild type or a variant sequence can exist together in a single cell, a condition known as mtDNA heteroplasmy. MtDNA single point mutations are typically detected by means of Next-Generation Sequencing (NGS) based on short reads which, however, are limited for the identification of structural mtDNA alterations. Recently, new NGS technologies based on long reads have been released, allowing to obtain sequences of several kilobases in length; this approach is suitable for detection of structural alterations affecting the mitochondrial genome. In the present work we illustrate the optimization of two sequencing protocols based on long-read Oxford Nanopore Technology to detect mtDNA structural alterations. This approach presents strong advantages in the analysis of mtDNA compared to both shortread NGS and traditional techniques, potentially becoming the method of choice for genetic studies on mtDNA. [ABSTRACT FROM AUTHOR]

Nocardia species do not replicate as rapidly as other pyogenic bacteria and nocardial infections can be highly fatal, particularly in immunocompromised patients. Here, we present the first report of fatal Nocardia kroppenstedtii bacteremic pneumonia and empyema thoracis diagnosed by next-generation sequencing (NGS) using the Oxford Nanopore Technologies' MinION device. The bacterium was not identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Due to its low equipment cost, short turn-around-time, and portable size, the Oxford Nanopore Technologies' MinION device is a useful platform for NGS in routine clinical microbiology laboratories.

Primary mitochondrial diseases are progressive genetic disorders affecting multiple organs and characterized by mitochondrial dysfunction. These disorders can be caused by mutations in nuclear genes coding proteins with mitochondrial localization or by genetic defects in the mitochondrial genome (mtDNA). The latter include point pathogenic variants and large-scale deletions/rearrangements. MtDNA molecules with the wild type or a variant sequence can exist together in a single cell, a condition known as mtDNA heteroplasmy. MtDNA single point mutations are typically detected by means of Next-Generation Sequencing (NGS) based on short reads which, however, are limited for the identification of structural mtDNA alterations. Recently, new NGS technologies based on long reads have been released, allowing to obtain sequences of several kilobases in length; this approach is suitable for detection of structural alterations affecting the mitochondrial genome. In the present work we illustrate the optimization of two sequencing protocols based on long-read Oxford Nanopore Technology to detect mtDNA structural alterations. This approach presents strong advantages in the analysis of mtDNA compared to both short-read NGS and traditional techniques, potentially becoming the method of choice for genetic studies on mtDNA.

Usutu virus (USUV) is a mosquito-borne zoonotic virus and one of the causes of flavivirus encephalitis in birds and occasionally in humans. USUV rapidly disperses in a susceptible host and vector environment, as is the case in South and Central Europe. However, compared to other flaviviruses, USUV has received less research attention and there is therefore limited access to whole-genome sequences and also to in-depth phylogenetic and phylodynamic analyses. To ease future molecular studies, this study compares first- (partial sequencing via Sanger), second- (Illumina), and third-generation (MinION Nanopore) sequencing platforms for USUV. With emphasis on MinION Nanopore sequencing, cDNA-direct and target-enrichment (amplicon-based) sequencing approaches were validated in parallel. The study was based on four samples from succumbed birds commonly collected throughout Germany. The samples were isolated from various sample matrices, organs as well as blood cruor, and included three different USUV lineages. We concluded that depending on the focus of a research project, amplicon-based MinION Nanopore sequencing can be an ideal cost- and time-effective alternative to Illumina in producing optimal genome coverage. It can be implemented for an array of lab- or field-based objectives, including among others: phylodynamic studies and the analysis of viral quasispecies. [ABSTRACT FROM AUTHOR]

BackgroundCoronavirus Disease-2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) accounts for considerable morbidity and mortality globally. Paucity of SARS-CoV-2 genetic data from Tanzania challenges in-country tracking of the pandemic. We sequenced SARS-CoV-2 isolated in the country to determine circulating strains, mutations and phylogenies and finally enrich international genetic databases especially with sequences from Africa.MethodsThis cross-sectional study utilized nasopharyngeal swabs of symptomatic and asymptomatic adults with positive polymerase chain reaction tests for COVID-19 from January to May 2021. Viral genomic libraries were prepared using ARTIC nCoV-2019 sequencing protocol version three. Whole-genome sequencing (WGS) was performed using Oxford Nanopore Technologies MinION device. In silico genomic data analysis was done on ARTIC pipeline version 1.2.1 using ARTIC nCoV-2019 bioinformatics protocol version 1.1.0.ResultsTwenty-nine (42%) out of 69 samples qualified for sequencing based on gel electrophoretic band intensity of multiplex PCR amplicons. Out of 29 isolates, 26 were variants of concern [Beta (n = 22); and Delta (n = 4)]. Other variants included Eta (n = 2) and B.1.530 (n = 1). We found combination of mutations (S: D80A, S: D215G, S: K417N, ORF3a: Q57H, E: P71L) in all Beta variants and absent in other lineages. The B.1.530 lineage carried mutations with very low cumulative global prevalence, these were nsp13:M233I, nsp14:S434G, ORF3a:A99S, S: T22I and S: N164H. The B.1.530 lineage clustered phylogenetically with isolates first reported in south-east Kenya, suggesting regional evolution of SARS-CoV-2.ConclusionWe provide evidence of existence of Beta, Delta, Eta variants and a locally evolving lineage (B.1.530) from samples collected in early 2021 in Tanzania. This work provides a model for ongoing WGS surveillance that will be required to inform on emerging and circulating SARS-CoV-2 diversity in Tanzania and East Africa.

Co-infections of avian species with different RNA viruses and pathogenic bacteria are often misdiagnosed or incompletely characterized using targeted diagnostic methods, which could affect the accurate management of clinical disease. A non-targeted sequencing approach with rapid and precise characterization of pathogens should help respiratory disease management by providing a comprehensive view of the causes of disease. Long-read portable sequencers have significant potential advantages over established short-read sequencers due to portability, speed, and lower cost. The applicability of short reads random sequencing for direct detection of pathogens in clinical poultry samples has been previously demonstrated. Here we demonstrate the feasibility of long read random sequencing approaches to identify disease agents in clinical samples. Experimental oropharyngeal swab samples (n = 12) from chickens infected with infectious bronchitis virus (IBV), avian influenza virus (AIV) and Mycoplasma synoviae (MS) and field-collected clinical oropharyngeal swab samples (n = 11) from Kenyan live bird markets previously testing positive for Newcastle disease virus (NDV) were randomly sequenced on the MinION platform and results validated by comparing to real time PCR and short read random sequencing in the Illumina MiSeq platform. In the swabs from experimental infections, each of three agents in every RT-qPCR-positive sample (Ct range 19–34) was detectable within 1 h on the MinION platform, except for AIV one agent in one sample (Ct = 36.21). Nine of 12 IBV-positive samples were assigned genotypes within 1 h, as were five of 11 AIV-positive samples. MinION relative abundances of the test agent (AIV, IBV and MS) were highly correlated with RT-qPCR Ct values (R range−0.82 to−0.98). In field-collected clinical swab samples, NDV (Ct range 12–37) was detected in all eleven samples within 1 h of MinION sequencing, with 10 of 11 samples accurately genotyped within 1 h. All NDV-positive field samples were found to be co-infected with one or more additional respiratory agents. These results demonstrate that MinION sequencing can provide rapid, and sensitive non-targeted detection and genetic characterization of co-existing respiratory pathogens in clinical samples with similar performance to the Illumina MiSeq.

Usutu virus (USUV) is a mosquito-borne zoonotic virus and one of the causes of flavivirus encephalitis in birds and occasionally in humans. USUV rapidly disperses in a susceptible host and vector environment, as is the case in South and Central Europe. However, compared to other flaviviruses, USUV has received less research attention and there is therefore limited access to whole-genome sequences and also to in-depth phylogenetic and phylodynamic analyses. To ease future molecular studies, this study compares first- (partial sequencing via Sanger), second- (Illumina), and third-generation (MinION Nanopore) sequencing platforms for USUV. With emphasis on MinION Nanopore sequencing, cDNA-direct and target-enrichment (amplicon-based) sequencing approaches were validated in parallel. The study was based on four samples from succumbed birds commonly collected throughout Germany. The samples were isolated from various sample matrices, organs as well as blood cruor, and included three different USUV lineages. We concluded that depending on the focus of a research project, amplicon-based MinION Nanopore sequencing can be an ideal cost- and time-effective alternative to Illumina in producing optimal genome coverage. It can be implemented for an array of lab- or field-based objectives, including among others: phylodynamic studies and the analysis of viral quasispecies.

As SARS-CoV-2 variants of concern emerged, the genome sequencing of SARS-CoV-2 strains became more important. In this study, SARS-CoV-2 was sequenced using amplicon-based genome sequencing with MinION. The primer panel used in this study consisted of only 11 primer panels and the size of the amplicons was approximately 3 kb. Full genome sequences were obtained with a hundred copies of the SARS-CoV-2 genome, and 92.33% and 75.39% of the genome sequences were obtained with 10 copies of the SARS-CoV-2 genome. The few differences in nucleotide sequences originated from mutations in laboratory cultures and/or mixed nucleotide sequences. The quantification of the SARS-CoV-2 genomic RNA was done using RT-ddPCR methods, and the level of LoD indicated that this sequencing method can be used for any RT-qPCR positive clinical sample. The sequencing results of the SARS-CoV-2 variants and clinical samples showed that our methods were very reliable. The genome sequences of five individual clinical samples were almost identical, and the analysis of the sequence variance showed that most of these nucleotide substitutions were observed in the genome sequences of the other clinical samples, indicating this amplicon-based whole-genome sequencing method can be used in various clinical fields. [ABSTRACT FROM AUTHOR]

Microbial pathogens may be present in different types of foods, and hence the development of novel methods to assure consumers’ safeness is of great interest. Molecular methods are known to provide sensitive and rapid results; however, they are typically targeted approaches. In recent years, the advent of non-targeted approaches based on next-generation sequencing (NGS) has emerged as a rational way to proceed. This technology allows for the detection of several pathogens simultaneously. Furthermore, with the same set of data, it is possible to characterize the microorganisms in terms of serotype, virulence, and/ or resistance genes, among other molecular features. In the current study, a novel method for the detection of Listeria monocytogenes based on the “quasimetagenomics” approach was developed. Different enrichment media and immunomagnetic separation (IMS) strategies were compared to determine the best approach in terms of L. monocytogenes sequences generated from smoked salmon samples. Finally, the data generated were analyzed with a user-friendly workflow that simultaneously provided the species identification, serotype, and antimicrobial resistance genes. The new method was thoroughly evaluated against a culture-based approach, using smoked salmon inoculated with L. monocytogenes as the matrix of choice. The sequencing method reached a very low limit of detection (LOD50, 1.2 CFU/ 25 g) along with high diagnostic sensitivity and specificity (100%), and a perfect correlation with the culture-based method (Cohen’s k = 1.00). Overall, the proposed method overcomes all the major limitations reported for the implementation of NGS as a routine food testing technology and paves the way for future developments taking its advantage into consideration. [ABSTRACT FROM AUTHOR]

Microbial pathogens may be present in different types of foods, and hence the development of novel methods to assure consumers' safeness is of great interest. Molecular methods are known to provide sensitive and rapid results; however, they are typically targeted approaches. In recent years, the advent of non-targeted approaches based on next-generation sequencing (NGS) has emerged as a rational way to proceed. This technology allows for the detection of several pathogens simultaneously. Furthermore, with the same set of data, it is possible to characterize the microorganisms in terms of serotype, virulence, and/ or resistance genes, among other molecular features. In the current study, a novel method for the detection of Listeria monocytogenes based on the “quasimetagenomics” approach was developed. Different enrichment media and immunomagnetic separation (IMS) strategies were compared to determine the best approach in terms of L. monocytogenes sequences generated from smoked salmon samples. Finally, the data generated were analyzed with a user-friendly workflow that simultaneously provided the species identification, serotype, and antimicrobial resistance genes. The new method was thoroughly evaluated against a culture-based approach, using smoked salmon inoculated with L. monocytogenes as the matrix of choice. The sequencing method reached a very low limit of detection (LOD50, 1.2 CFU/ 25 g) along with high diagnostic sensitivity and specificity (100%), and a perfect correlation with the culture-based method (Cohen's k = 1.00). Overall, the proposed method overcomes all the major limitations reported for the implementation of NGS as a routine food testing technology and paves the way for future developments taking its advantage into consideration.

During the last decades, antimicrobial resistance (AMR) has become a global public health concern. Nowadays multi-drug resistance is commonly observed in strains of Vibrio cholerae , the etiological agent of cholera. In order to limit the spread of pathogenic drug-resistant bacteria and to maintain treatment options the analysis of clinical samples and their AMR profiles are essential. Particularly, in low-resource settings a timely analysis of AMR profiles is often impaired due to lengthy culturing procedures for antibiotic susceptibility testing or lack of laboratory capacity. In this study, we explore the applicability of whole genome sequencing for the prediction of AMR profiles of V. cholerae. We developed the pipeline CholerAegon for the in silico prediction of AMR profiles of 82 V. cholerae genomes assembled from long and short sequencing reads. By correlating the predicted profiles with results from phenotypic antibiotic susceptibility testing we show that the prediction can replace in vitro susceptibility testing for five of seven antibiotics. Because of the relatively low costs, possibility for real-time data analyses, and portability, the Oxford Nanopore Technologies MinION sequencing platform—especially in light of an upcoming less error-prone technology for the platform—appears to be well suited for pathogen genomic analyses such as the one described here. Together with CholerAegon, it can leverage pathogen genomics to improve disease surveillance and to control further spread of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

During the last decades, antimicrobial resistance (AMR) has become a global public health concern. Nowadays multi-drug resistance is commonly observed in strains of Vibrio cholerae, the etiological agent of cholera. In order to limit the spread of pathogenic drug-resistant bacteria and to maintain treatment options the analysis of clinical samples and their AMR profiles are essential. Particularly, in low-resource settings a timely analysis of AMR profiles is often impaired due to lengthy culturing procedures for antibiotic susceptibility testing or lack of laboratory capacity. In this study, we explore the applicability of whole genome sequencing for the prediction of AMR profiles of V. cholerae. We developed the pipeline CholerAegon for the in silico prediction of AMR profiles of 82 V. cholerae genomes assembled from long and short sequencing reads. By correlating the predicted profiles with results from phenotypic antibiotic susceptibility testing we show that the prediction can replace in vitro susceptibility testing for five of seven antibiotics. Because of the relatively low costs, possibility for real-time data analyses, and portability, the Oxford Nanopore Technologies MinION sequencing platform—especially in light of an upcoming less error-prone technology for the platform—appears to be well suited for pathogen genomic analyses such as the one described here. Together with CholerAegon, it can leverage pathogen genomics to improve disease surveillance and to control further spread of antimicrobial resistance.

Marine plastic debris serve as substrates for the colonization of a variety of prokaryote and eukaryote organisms. Of particular interest are the microorganisms that have adapted to thrive on plastic as they may contain genes, enzymes or pathways involved in the adhesion or metabolism of plastics. We implemented DNA metabarcoding with nanopore MinION sequencing to compare the 1-month-old biomes of hydrolyzable (polyethylene terephthalate) and non-hydrolyzable (polyethylene) plastics surfaces vs. those of glass and the surrounding water in a Mediterranean Sea marina. We sequenced longer 16S rRNA, 18S rRNA, and ITS barcode loci for a more comprehensive taxonomic profiling of the bacterial, protist, and fungal communities, respectively. Long read sequencing enabled high-resolution mapping to genera and species. Using previously established methods we performed differential abundance screening and identified 30 bacteria and five eukaryotic species, that were differentially abundant on plastic compared to glass. This approach will allow future studies to characterize the plastisphere communities and to screen for microorganisms with a plastic-metabolism potential. [ABSTRACT FROM AUTHOR]

Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing. [ABSTRACT FROM AUTHOR]

The Illumina MiSeq platform has been widely used as a standard method for studying the rumen microbiota. However, the low resolution of taxonomic identification is the only disadvantage of MiSeq amplicon sequencing, as it targets a part of the 16S rRNA gene. In the present study, we performed three experiments to establish a high-resolution and high-throughput rumen microbial profiling approach using a combination of MinION platform and buccal swab sample, which is a proxy for rumen contents. In experiment 1, rumen contents and buccal swab samples were collected simultaneously from cannulated cattle (n = 6) and used for microbiota analysis using three different analytical workflows: amplicon sequencing of the V3–V4 region of the 16S rRNA gene using MiSeq and amplicon sequencing of near full-length 16S rRNA gene using MinION or PacBio Sequel II. All reads derived from the MinION and PacBio platforms were classified at the species-level. In experiment 2, rumen fluid samples were collected from beef cattle (n = 28) and used for 16S rRNA gene amplicon sequencing using the MinION platform to evaluate this sequencing platform for rumen microbiota analysis. We confirmed that the MinION platform allowed species-level taxa assignment for the predominant bacterial groups, which were previously identified at the family- and genus-level using the MiSeq platform. In experiment 3, buccal swab samples were collected from beef cattle (n = 30) and used for 16S rRNA gene amplicon sequencing using the MinION platform to validate the applicability of a combination of the MinION platform and buccal swab samples for rumen microbiota analysis. The distribution of predominant bacterial taxa in the buccal swab samples was similar to that in the rumen samples observed in experiment 2. Based on these results, we concluded that the combination of the MinION platform and buccal swab samples may be potentially applied for rumen microbial analysis in large-scale studies. [ABSTRACT FROM AUTHOR]

Long-read sequencing (LRS) can resolve repetitive regions, a limitation of short read (SR) data. Reduced cost and instrument size has led to a steady increase in LRS across diagnostics and research. Here, we re-basecalled FAST5 data sequenced between 2018 and 2021 and analyzed the data in relation to gDNA across a large dataset (n = 200) spanning a wide GC content (25–67%). We examined whether re-basecalled data would improve the hybrid assembly, and, for a smaller cohort, compared long read (LR) assemblies in the context of antimicrobial resistance (AMR) genes and mobile genetic elements. We included a cost analysis when comparing SR and LR instruments. We compared the R9 and R10 chemistries and reported not only a larger yield but increased read quality with R9 flow cells. There were often discrepancies with ARG presence/absence and/or variant detection in LR assemblies. Flye-based assemblies were generally efficient at detecting the presence of ARG on both the chromosome and plasmids. Raven performed more quickly but inconsistently recovered small plasmids, notably a ∼15-kb Col-like plasmid harboring bla KPC . Canu assemblies were the most fragmented, with genome sizes larger than expected. LR assemblies failed to consistently determine multiple copies of the same ARG as identified by the Unicycler reference. Even with improvements to ONT chemistry and basecalling, long-read assemblies can lead to misinterpretation of data. If LR data are currently being relied upon, it is necessary to perform multiple assemblies, although this is resource (computing) intensive and not yet readily available/useable. [ABSTRACT FROM AUTHOR]

Marine plastic debris serve as substrates for the colonization of a variety of prokaryote and eukaryote organisms. Of particular interest are the microorganisms that have adapted to thrive on plastic as they may contain genes, enzymes or pathways involved in the adhesion or metabolism of plastics. We implemented DNA metabarcoding with nanopore MinION sequencing to compare the 1-month-old biomes of hydrolyzable (polyethylene terephthalate) and non-hydrolyzable (polyethylene) plastics surfaces vs. those of glass and the surrounding water in a Mediterranean Sea marina. We sequenced longer 16S rRNA, 18S rRNA, and ITS barcode loci for a more comprehensive taxonomic profiling of the bacterial, protist, and fungal communities, respectively. Long read sequencing enabled high-resolution mapping to genera and species. Using previously established methods we performed differential abundance screening and identified 30 bacteria and five eukaryotic species, that were differentially abundant on plastic compared to glass. This approach will allow future studies to characterize the plastisphere communities and to screen for microorganisms with a plastic-metabolism potential.

Long-read sequencing (LRS) can resolve repetitive regions, a limitation of short read (SR) data. Reduced cost and instrument size has led to a steady increase in LRS across diagnostics and research. Here, we re-basecalled FAST5 data sequenced between 2018 and 2021 and analyzed the data in relation to gDNA across a large dataset (n = 200) spanning a wide GC content (25–67%). We examined whether re-basecalled data would improve the hybrid assembly, and, for a smaller cohort, compared long read (LR) assemblies in the context of antimicrobial resistance (AMR) genes and mobile genetic elements. We included a cost analysis when comparing SR and LR instruments. We compared the R9 and R10 chemistries and reported not only a larger yield but increased read quality with R9 flow cells. There were often discrepancies with ARG presence/absence and/or variant detection in LR assemblies. Flye-based assemblies were generally efficient at detecting the presence of ARG on both the chromosome and plasmids. Raven performed more quickly but inconsistently recovered small plasmids, notably a ∼15-kb Col-like plasmid harboring blaKPC. Canu assemblies were the most fragmented, with genome sizes larger than expected. LR assemblies failed to consistently determine multiple copies of the same ARG as identified by the Unicycler reference. Even with improvements to ONT chemistry and basecalling, long-read assemblies can lead to misinterpretation of data. If LR data are currently being relied upon, it is necessary to perform multiple assemblies, although this is resource (computing) intensive and not yet readily available/useable.

The Illumina MiSeq platform has been widely used as a standard method for studying the rumen microbiota. However, the low resolution of taxonomic identification is the only disadvantage of MiSeq amplicon sequencing, as it targets a part of the 16S rRNA gene. In the present study, we performed three experiments to establish a high-resolution and high-throughput rumen microbial profiling approach using a combination of MinION platform and buccal swab sample, which is a proxy for rumen contents. In experiment 1, rumen contents and buccal swab samples were collected simultaneously from cannulated cattle (n = 6) and used for microbiota analysis using three different analytical workflows: amplicon sequencing of the V3–V4 region of the 16S rRNA gene using MiSeq and amplicon sequencing of near full-length 16S rRNA gene using MinION or PacBio Sequel II. All reads derived from the MinION and PacBio platforms were classified at the species-level. In experiment 2, rumen fluid samples were collected from beef cattle (n = 28) and used for 16S rRNA gene amplicon sequencing using the MinION platform to evaluate this sequencing platform for rumen microbiota analysis. We confirmed that the MinION platform allowed species-level taxa assignment for the predominant bacterial groups, which were previously identified at the family- and genus-level using the MiSeq platform. In experiment 3, buccal swab samples were collected from beef cattle (n = 30) and used for 16S rRNA gene amplicon sequencing using the MinION platform to validate the applicability of a combination of the MinION platform and buccal swab samples for rumen microbiota analysis. The distribution of predominant bacterial taxa in the buccal swab samples was similar to that in the rumen samples observed in experiment 2. Based on these results, we concluded that the combination of the MinION platform and buccal swab samples may be potentially applied for rumen microbial analysis in large-scale studies.

Molecular typing is an essential tool that has been extensively applied in laboratories as well as in clinical settings. Next-generation sequencing technologies promise high-throughput and cost-effective molecular applications; however, the accessibility of these technologies is limited due to the high capital cost. Oxford Nanopore Technologies (ONT) offers a MinION device with the advantages of real-time data analysis, rapid library preparation, and low cost per test. However, the advantages of the MinION device are often overshadowed by its lower raw accuracy. Herein, we present a concise multilocus sequence typing protocol of Staphylococcus aureus using multiplex polymerase chain reaction and Rapid Barcoding Kit for barcoding and MinION device for sequencing. Moreover, to clarify the effects of carryover DNA on tasks that require high sequence accuracy, we used the MinION flow cell in successive runs of washing and reusing. Our results revealed that the MinION flow cell could achieve accurate typing of a total of 467 samples with 3,269 kilobase-long genes within a total of 5 runs. This thus demonstrates the effectiveness of a portable nanopore MinION sequencer in providing accurate, rapid, and routine molecular typing.

We present the first report of histology- and culture-proven Mycobacterium marinum infection diagnosed by next-generation sequencing (NGS). It took

Background: Mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2) represent a major genetic cause of pulmonary arterial hypertension (PAH). Identification of BMPR2 mutations is crucial for the genetic diagnosis of PAH. MinION nanopore sequencer is a portable third-generation technology that enables long-read sequencing at a low-cost. This nanopore technology-based device has not been used previously for PAH diagnosis. This study aimed to determine the feasibility of using MinION nanopore sequencing for the genetic analysis of PAH patients, focused on BMPR2.Methods: We developed a protocol for the custom bioinformatics pipeline analysis of long reads generated by long-PCR. To evaluate the potential of using MinION sequencing in PAH, we analyzed five samples, including those of two idiopathic PAH patients and a family of three members with one affected patient. Sanger sequencing analysis was performed to validate the variants.Results: The median read length was around 3.4 kb and a good mean quality score of approximately 19 was obtained. The total number of reads generated was uniform among the cases and ranged from 2,268,263 to 3,126,719. The coverage was consistent across flow cells in which the average number of reads per base ranged from 80,375 to 135,603. We identified two polymorphic variants and three mutations in four out of five patients. Certain indel variant calling-related errors were observed, mostly outside coding sequences.Conclusion: We have shown the ability of this portable nanopore sequencer to detect BMPR2 mutations in patients with PAH. The MinION nanopore sequencer is a promising tool for screening BMPR2 mutations, especially in small laboratories and research groups.

Blastocystis is frequently reported in fecal samples from animals and humans worldwide, and a variety of subtypes (STs) have been observed in wild and domestic animals. In Colombia, few studies have focused on the transmission dynamics and epidemiological importance of Blastocystis in animals. In this study, we characterized the frequency and subtypes of Blastocystis in fecal samples of domestic animals including pigs, minipigs, cows, dogs, horses, goats, sheep, and llama from three departments of Colombia. Of the 118 fecal samples included in this study 81.4% (n = 96) were positive for Blastocystis using a PCR that amplifies a fragment of the small subunit ribosomal RNA (SSU rRNA) gene. PCR positive samples were sequenced by next generation amplicon sequencing (NGS) to determine subtypes. Eleven subtypes were detected, ten previously reported, ST5 (50.7%), ST10 (47.8%), ST25 (34.3%), ST26 (29.8%), ST21 (22.4%), ST23 (22.4%), ST1 (17.9%), ST14 (16.4%), ST24 (14.9%), ST3 (7.5%), and a novel subtype, named ST32 (3.0%). Mixed infection and/or intra -subtype variations were identified in most of the samples. Novel ST32 was observed in two samples from a goat and a cow. To support novel subtype designation, a MinION based sequencing strategy was used to generate the full-length of the SSU rRNA gene. Comparison of full-length nucleotide sequences with those from current valid subtypes supported the designation of ST32. This is the first study in Colombia using NGS to molecularly characterize subtypes of Blastocystis in farm animals. A great diversity of subtypes was observed in domestic animals including subtypes previously identified in humans. Additionally, subtype overlap between the different hosts examined in this study were observed. These findings highlight the presence of Blastocystis subtypes with zoonotic potential in farm animals indicating that farm animals could play a role in transmission to humans.

Since the emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in December 2019, the scientific community has been sharing data on epidemiology, diagnostic methods, and whole-genomic sequences almost in real time. The latter have already facilitated phylogenetic analyses, transmission chain tracking, protein modeling, the identification of possible therapeutic targets, timely risk assessment, and identification of novel variants. We have established and evaluated an amplification-based approach for whole-genome sequencing of SARS-CoV-2. It can be used on the miniature-sized and field-deployable sequencing device Oxford Nanopore MinION, with sequencing library preparation time of 10 min. We show that the generation of 50,000 total reads per sample is sufficient for a near complete coverage (>90%) of the SARS-CoV-2 genome directly from patient samples even if virus concentration is low (Ct 35, corresponding to approximately 5 genome copies per reaction). For patient samples with high viral load (Ct 18–24), generation of 50,000 reads in 1–2 h was shown to be sufficient for a genome coverage of >90%. Comparison to Illumina data reveals an accuracy that suffices to identify virus mutants. AmpliCoV can be applied whenever sequence information on SARS-CoV-2 is required rapidly, for instance for the identification of circulating virus mutants.

Algal blooms occur in freshwater bodies throughout the world, often leading to fish kills. Cases of these kills along the Ural River were reported in 2018–2019, involving significant amount of sturgeon in fish farming areas. In this study, the analysis of algal samples from the delta of the Ural River up to 100 km inland was carried out from August to December 2019 using imaging flow cytometry (IFC), molecular biological, and microscopic techniques. We identified the filamentous cyanobacteria Cuspidothrix issatschenkoi, Dolichospermum cf. flos-aquae, Dolichospermum cf. macrosporum, Pseudanabaena limnetica, and Planktothrix spp. as the dominant potentially toxic phytoplankton species, and we also found minor quantities of Cylindrospermopsis raciborskii. For the first time, molecular phylogenetic investigations of field clones of cyanobacteria from Ural River were carried out to establish the taxa of the dominant species and to identify the presence of genes encoding toxins. The complementary analysis with nanopore-based next-generation sequencing overlapped with the results of IFC and was instrumental in revealing minor cyanobacteria taxa. Real-time PCR analysis and sequencing indicated the presence of Microcystis and ADA-clade spp. as well as genes associated with the production of microcystin (mcyE) and the algal neurotoxin saxitoxin (sxtA) originating from cyanobacteria. These findings suggest that toxin-producing cyanobacteria could become a threat in the Ural River near Atyrau, which can significantly affect aquaculture in the region.

The sequencing of viral genomes provides important data for the prevention and control of foot-and-mouth disease (FMD) outbreaks. Sequence data can be used for strain identification, outbreak tracing, and aiding the selection of the most appropriate vaccine for the circulating strains. At present, sequencing of FMD virus (FMDV) relies upon the time-consuming transport of samples to well-resourced laboratories. The Oxford Nanopore Technologies' MinION portable sequencer has the potential to allow sequencing in remote, decentralised laboratories closer to the outbreak location. In this study, we investigated the utility of the MinION to generate sequence data of sufficient quantity and quality for the characterisation of FMDV serotypes O, A, Asia 1. Prior to sequencing, a universal two-step RT-PCR was used to amplify parts of the 5′UTR, as well as the leader, capsid and parts of the 2A encoding regions of FMDV RNA extracted from three sample matrices: cell culture supernatant, tongue epithelial suspension and oral swabs. The resulting consensus sequences were compared with reference sequences generated on the Illumina MiSeq platform. Consensus sequences with an accuracy of 100% were achieved within 10 and 30 min from the start of the sequencing run when using RNA extracted from cell culture supernatants and tongue epithelial suspensions, respectively. In contrast, sequencing from swabs required up to 2.5 h. Together these results demonstrated that the MinION sequencer can be used to accurately and rapidly characterise serotypes A, O, and Asia 1 of FMDV using amplicons amplified from a variety of different sample matrices.

Since the emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in December 2019, the scientific community has been sharing data on epidemiology, diagnostic methods, and whole-genomic sequences almost in real time. The latter have already facilitated phylogenetic analyses, transmission chain tracking, protein modeling, the identification of possible therapeutic targets, timely risk assessment, and identification of novel variants. We have established and evaluated an amplification-based approach for whole-genome sequencing of SARS-CoV-2. It can be used on the miniature-sized and field-deployable sequencing device Oxford Nanopore MinION, with sequencing library preparation time of 10 min. We show that the generation of 50,000 total reads per sample is sufficient for a near complete coverage (>90%) of the SARS-CoV-2 genome directly from patient samples even if virus concentration is low (Ct 35, corresponding to approximately 5 genome copies per reaction). For patient samples with high viral load (Ct 18–24), generation of 50,000 reads in 1–2 h was shown to be sufficient for a genome coverage of >90%. Comparison to Illumina data reveals an accuracy that suffices to identify virus mutants. AmpliCoV can be applied whenever sequence information on SARS-CoV-2 is required rapidly, for instance for the identification of circulating virus mutants. [ABSTRACT FROM AUTHOR]

Malaria is responsible for unacceptably high morbidity and mortality, especially in Sub-Saharan African Nations. Malaria is caused by member species’ of the genus Plasmodium and despite concerted and at times valiant efforts, the underlying pathophysiological processes leading to severe disease are poorly understood. Here we describe zoonotic malaria caused by Plasmodium knowlesi and the utility of this parasite as a model system for severe malaria. We present a method to generate long-read third-generation Plasmodium genome sequence data from archived clinical samples using the MinION platform. The method and technology are accessible, affordable and data is generated in real-time. We propose that by widely adopting this methodology important information on clinically relevant parasite diversity, including multiple gene family members, from geographically distinct study sites will emerge. Our goal, over time, is to exploit the duality of P. knowlesi as a well-used laboratory model and human pathogen to develop a representative translational model system for severe malaria that is informed by clinically relevant parasite diversity.

Identification of human leukocyte antigen (HLA) alleles from next-generation sequencing (NGS) data is challenging because of the high polymorphism and mosaic nature of HLA genes. Owing to the complex nature of HLA genes and consequent challenges in allele assignment, Oxford Nanopore Technologies’ (ONT) single-molecule sequencing technology has been of great interest due to its fitness for sequencing long reads. In addition to the read length, ONT’s advantages are its portability and possibility for a rapid real-time sequencing, which enables a simultaneous data analysis. Here, we describe a targeted RNA-based method for HLA typing using ONT sequencing and SeqNext-HLA SeqPilot software (JSI Medical Systems GmbH). Twelve classical HLA genes were enriched from cDNA of 50 individuals, barcoded, pooled, and sequenced in 10 MinION R9.4 SpotON flow cell runs producing over 30,000 reads per sample. Using barcoded 2D reads, SeqPilot assigned HLA alleles to two-field typing resolution or higher with the average read depth of 1750x. Sequence analysis resulted in 99–100% accuracy at low-resolution level (one-field) and in 74–100% accuracy at high-resolution level (two-field) with the expected alleles. There are still some limitations with ONT RNA sequencing, such as noisy reads, homopolymer errors, and the lack of robust algorithms, which interfere with confident allele assignment. These issues need to be inspected carefully in the future to improve the allele call rates. Nevertheless, here we show that sequencing of multiplexed cDNA amplicon libraries on ONT MinION can produce accurate high-resolution typing results of 12 classical HLA loci. For HLA research, ONT RNA sequencing is a promising method due to its capability to sequence full-length HLA transcripts. In addition to HLA genotyping, the technique could also be applied for simultaneous expression analysis.

Identification of human leukocyte antigen (HLA) alleles from next-generation sequencing (NGS) data is challenging because of the high polymorphism and mosaic nature of HLA genes. Owing to the complex nature of HLA genes and consequent challenges in allele assignment, Oxford Nanopore Technologies' (ONT) single-molecule sequencing technology has been of great interest due to its fitness for sequencing long reads. In addition to the read length, ONT's advantages are its portability and possibility for a rapid real-time sequencing, which enables a simultaneous data analysis. Here, we describe a targeted RNA-based method for HLA typing using ONT sequencing and SeqNext-HLA SeqPilot software (JSI Medical Systems GmbH). Twelve classical HLA genes were enriched from cDNA of 50 individuals, barcoded, pooled, and sequenced in 10 MinION R9.4 SpotON flow cell runs producing over 30,000 reads per sample. Using barcoded 2D reads, SeqPilot assigned HLA alleles to two-field typing resolution or higher with the average read depth of 1750x. Sequence analysis resulted in 99–100% accuracy at low-resolution level (one-field) and in 74–100% accuracy at high-resolution level (two-field) with the expected alleles. There are still some limitations with ONT RNA sequencing, such as noisy reads, homopolymer errors, and the lack of robust algorithms, which interfere with confident allele assignment. These issues need to be inspected carefully in the future to improve the allele call rates. Nevertheless, here we show that sequencing of multiplexed cDNA amplicon libraries on ONT MinION can produce accurate high-resolution typing results of 12 classical HLA loci. For HLA research, ONT RNA sequencing is a promising method due to its capability to sequence full-length HLA transcripts. In addition to HLA genotyping, the technique could also be applied for simultaneous expression analysis. [ABSTRACT FROM AUTHOR]

Malaria is responsible for unacceptably high morbidity and mortality, especially in Sub-Saharan African Nations. Malaria is caused by member species' of the genus Plasmodium and despite concerted and at times valiant efforts, the underlying pathophysiological processes leading to severe disease are poorly understood. Here we describe zoonotic malaria caused by Plasmodium knowlesi and the utility of this parasite as a model system for severe malaria. We present a method to generate long-read third-generation Plasmodium genome sequence data from archived clinical samples using the MinION platform. The method and technology are accessible, affordable and data is generated in real-time. We propose that by widely adopting this methodology important information on clinically relevant parasite diversity, including multiple gene family members, from geographically distinct study sites will emerge. Our goal, over time, is to exploit the duality of P. knowlesi as a well-used laboratory model and human pathogen to develop a representative translational model system for severe malaria that is informed by clinically relevant parasite diversity. [ABSTRACT FROM AUTHOR]

Molecular characterization of genetically modified plants can provide crucial information for the development of detection and identification methods, to comply with traceability, and labeling requirements prior to commercialization. Detailed description of the genetic modification was previously a challenging step in the safety assessment, since it required the use of laborious and time-consuming techniques. In this study an accurate, simple, and fast method was developed for molecular characterization of genetically modified (GM) plants, following a user-friendly workflow for researchers with limited bioinformatic capabilities. Three GM events from a diverse array of crop species—perennial ryegrass, white clover, and canola—were used to test the approach that exploits long-read sequencing by the MinION device, from Oxford Nanopore Technologies. The method delivered a higher degree of resolution of the transgenic events within the host genome than has previously been possible with the standard Illumina short-range sequencing strategies. The flanking sequences, copy number, and presence of backbone sequences, and overall transgene insertion structure were determined for each of the plant genomes, with the additional identification of moderate-sized secondary insertions that would have previously been missed. The proposed workflow takes only about 1 week from DNA extraction to analyzed result, and the method will complement the existing approaches for molecular characterization of GM plants, since it makes the process faster, simpler, and more cost-effective. [ABSTRACT FROM AUTHOR]

Whole-genome sequencing (WGS) of viruses from patient or environmental samples can provide tremendous insight into the epidemiology, drug resistance or evolution of a virus. However, we face two common hurdles in obtaining robust sequence information; the low copy number of viral genomes in specimens and the error introduced by WGS techniques. To optimize detection and minimize error in WGS of hantaviruses, we tested four amplification approaches and different amplicon pooling methods for library preparation and examined these preparations using two sequencing platforms, Illumina MiSeq and Oxford Nanopore Technologies MinION. First, we tested and optimized primers used for whole segment PCR or one kilobase amplicon amplification for even coverage using RNA isolated from the supernatant of virus-infected cells. Once optimized we assessed two sources of total RNA, virus-infected cells and supernatant from the virus-infected cells, with four variations of primer pooling for amplicons, and six different amplification approaches. We show that 99–100% genome coverage was obtained using a one-step RT-PCR reaction with one forward and reverse primer. Using a two-step RT-PCR with three distinct tiling approaches for the three genomic segments (vRNAs), we optimized primer pooling approaches for PCR amplification to achieve a greater number of aligned reads, average depth of genome, and genome coverage. The single nucleotide polymorphisms identified from MiSeq and MinION sequencing suggested intrinsic mutation frequencies of ~10−5-10−7 per genome and 10−4-10−5 per genome, respectively. We noted no difference in the coverage or accuracy when comparing WGS results with amplicons amplified from RNA extracted from infected cells or supernatant of these infected cells. Our results show that high-throughput diagnostics requiring the identification of hantavirus species or strains can be performed using MiSeq or MinION using a one-step approach. However, the two-step MiSeq approach outperformed the MinION in coverage depth and accuracy, and hence would be superior for assessment of genomes for epidemiology or evolutionary questions using the methods developed herein.

Peste-des-petits-ruminants virus (PPRV) is currently the focus of a control and eradication program. Full genome sequencing has the opportunity to become a powerful tool in the eradication program by improving molecular epidemiology and the study of viral evolution. PPRV is prevalent in many resource-constrained areas, with long distances to laboratory facilities, which can lack the correct equipment for high-throughput sequencing. Here we present a protocol for near full or full genome sequencing of PPRV. The use of a portable miniPCR and MinION brings the laboratory to the field and in addition makes the production of a full genome possible within 24 h of sampling. The protocol has been successfully used on virus isolates from cell cultures and field isolates from tissue samples of naturally infected goats.

Global human health is increasingly challenged by emerging viral threats, especially those observed over the last 20 years with coronavirus-related human diseases, such as the Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS). Recently, in late December 2019, a novel Betacoronavirus, SARS-CoV-2, originating from the Chinese city of Wuhan, emerged and was then identified as the causative agent of a new severe form of pneumonia, COVID-19. Real-time genome sequencing in such viral outbreaks is a key issue to confirm identification and characterization of the involved pathogen and to help establish public health measures. Here, we implemented an amplicon-based sequencing approach combined with easily deployable next-generation sequencers, the small and hand-held MinION sequencer and the latest most compact Illumina sequencer, the iSeq100TM system. Our results highlighted the great potential of the amplicon-based approach to obtain consensus genomes of SARS-CoV-2 from clinical samples in just a few hours. Both these mobile next-generation sequencers are proven to be efficient to obtain viral sequences and easy to implement, with a minimal laboratory environment requirement, providing useful opportunities in the field and in remote areas.

The presence of extended-spectrum β-lactamase (ESBL)-producing bacteria in environmental sources has been reported worldwide and constitutes a serious risk of community-acquired infections with limited treatment options. The current study aimed to explore the presence of these worrisome bacteria in a pond located at the Norwegian University of Life Sciences in Ås, Norway. A total of 98 bacterial isolates survived growth on selective chromogenic media and were identified by 16S rRNA Sanger sequencing. All strains were evaluated for the presence of the most commonly found β-lactamases and ESBLs in clinical settings (blaCTX–M groups 1, 2, and 9, blaCMY, blaSHV, and blaTEM) and carbapenemases (blaIMP, blaKPC, blaNDM, blaOXA, blaSFC1, blaVIM) through multiplex PCR. A total of eight strains were determined to contain one or more genes of interest. Phenotypic resistance to 18 antimicrobial agents was assessed and isolates were subjected to whole genome sequencing through a combination of Oxford Nanopore’s MinION and Illumina’s MiSeq. Results revealed the presence of β-lactamase and ESBL-producing Escherichia coli, Klebsiella pneumoniae, Stenotrophomonas maltophilia, and a Paraburkholderia spp. Identified β-lactamases and ESBLs include blaCTX–M, blaTEM, blaCMY, blaSHV and a possible blaKPC-like gene, with both documented and novel sequences established. In addition, two inducible β-lactamases were found, a class A β-lactamase (L1) and a cephalosporinase (L2). All strains were determined to be multidrug resistant and numerous resistance genes to non-β-lactams were observed. In conclusion, this study demonstrates that environmental sources are a potential reservoir of clinically relevant ESBL-producing bacteria that may pose a health risk to humans upon exposure.

Harmful algal blooms (HABs) are a naturally occurring global phenomena that have the potential to impact fisheries, leisure and ecosystems, as well as posing a significant hazard to animal and human health. There is significant interest in the development and application of methodologies to study all aspects of the causative organisms and toxins associated with these events. This paper reports the first application of nanopore sequencing technology for the detection of eukaryotic harmful algal bloom organisms. The MinION sequencing platform from Oxford Nanopore technologies provides long read sequencing capabilities in a compact, low cost, and portable format. In this study we used the MinION to sequence long-range PCR amplicons from multiple dinoflagellate species with a focus on the genus Alexandrium. Primers applicable to a wide range of dinoflagellates were selected, meaning that although the study was primarily focused on Alexandrium the applicability to three additional genera of toxic algae, namely; Gonyaulax, Prorocentrum, and Lingulodinium was also demonstrated. The amplicon generated here spanned approximately 3 kb of the rDNA cassette, including most of the 18S, the complete ITS1, 5.8S, ITS2 and regions D1 and D2 of the 28S. The inclusion of barcode genes as well as highly conserved regions resulted in identification of organisms to the species level. The analysis of reference cultures resulted in over 99% of all sequences being attributed to the correct species with an average identity above 95% from a reference list of over 200 species (see Supplementary Material 1). The use of mock community analysis within environmental samples highlighted that complex matrices did not prevent the ability to distinguish between phylogenetically similar species. Successful identification of causative organisms in environmental samples during natural toxic events further highlighted the potential of the assay. This study proves the suitability of nanopore sequencing technology for taxonomic identification of harmful algal bloom organisms and acquisition of data relevant to the World Health Organisations “one health” approach to marine monitoring.

Since human and mouse Y chromosomes contain repeated sequences, it is difficult to determine the precise sequences and analyze the function of individual Y chromosome genes. Therefore, the causes of many diseases and abnormalities related to Y chromosome genes, such as male infertility, remain unclear. In this study, to elucidate the mouse Y chromosome, we enriched the mouse Y chromosome using a fluorescence-activated cell sorter (FACS) equipped with commonly used UV and blue 488 nm lasers and read the nucleotides using the Oxford Nanopore MinION long-read sequencer. This sequencing strategy allows us to cover the whole known region as well as the potential undetermined region of the Y chromosome. FACS-based chromosome enrichment and long-read sequencing are suitable for analysis of the Y chromosome sequences and may lead to further understanding of the physiological role of Y chromosome genes.

Many nematode species are parasitic and threaten the health of plants and animals, including humans, on a global scale. Advances in DNA sequencing techniques have allowed for the rapid and accurate identification of many organisms including nematodes. However, the steps taken from sample collection in the field to molecular analysis and identification can take many days and depend on access to both immovable equipment and a specialized laboratory. Here, we present a protocol to genetically identify nematodes using 18S SSU rRNA sequencing using the MinION, a portable third generation sequencer, and proof that it is possible to perform all the molecular preparations on a fully portable molecular biology lab – the Bentolab. We show that both parasitic and free-living nematode species (Anisakis simplex, Panagrellus redivivus, Turbatrix aceti, and Caenorhabditis elegans) can be identified with a 96–100% accuracy compared to Sanger sequencing, requiring only 10–15 min of sequencing. This protocol is an essential first step toward genetically identifying nematodes in the field from complex natural environments (such as feces, soil, or marine sediments). This increased accessibility could in turn improve global information of nematode presence and distribution, aiding near-real-time global biomonitoring.

Whole-genome sequencing (WGS) of viruses from patient or environmental samples can provide tremendous insight into the epidemiology, drug resistance or evolution of a virus. However, we face two common hurdles in obtaining robust sequence information; the low copy number of viral genomes in specimens and the error introduced by WGS techniques. To optimize detection and minimize error in WGS of hantaviruses, we tested four amplification approaches and different amplicon pooling methods for library preparation and examined these preparations using two sequencing platforms, Illumina MiSeq and Oxford Nanopore Technologies MinION. First, we tested and optimized primers used for whole segment PCR or one kilobase amplicon amplification for even coverage using RNA isolated from the supernatant of virus-infected cells. Once optimized we assessed two sources of total RNA, virus-infected cells and supernatant from the virus-infected cells, with four variations of primer pooling for amplicons, and six different amplification approaches. We show that 99–100% genome coverage was obtained using a one-step RT-PCR reaction with one forward and reverse primer. Using a two-step RT-PCR with three distinct tiling approaches for the three genomic segments (vRNAs), we optimized primer pooling approaches for PCR amplification to achieve a greater number of aligned reads, average depth of genome, and genome coverage. The single nucleotide polymorphisms identified from MiSeq and MinION sequencing suggested intrinsic mutation frequencies of ~10−5-10−7 per genome and 10−4-10−5 per genome, respectively. We noted no difference in the coverage or accuracy when comparing WGS results with amplicons amplified from RNA extracted from infected cells or supernatant of these infected cells. Our results show that high-throughput diagnostics requiring the identification of hantavirus species or strains can be performed using MiSeq or MinION using a one-step approach. However, the two-step MiSeq approach outperformed the MinION in coverage depth and accuracy, and hence would be superior for assessment of genomes for epidemiology or evolutionary questions using the methods developed herein. [ABSTRACT FROM AUTHOR]