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West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

Methicillin-resistant Staphylococcus aureus (MRSA) is a variant of the more common Methicillin-Susceptible Staphylococcus aureus (MSSA), an opportunistic pathogen a portion of the human population carries as normal bacterial flora. When an outbreak of MRSA occurs, it is often important to determine if and how these strains are related to each other. In this report two different types of epidemiological methods were combined (namely Multi-Locus Sequence Typing and staphylococcal protein A-typing), in order to reduce workload, costs and time. A panel of resistance and virulence markers was also added to gather as much information about the culture as possible in a single analysis. To test the viability of the method extracted DNA and heat-treated bacterial cultures of both MRSA and MSSA were amplified with a curated panel of primers. These products were later sequenced with Nanopore’s MinION using the Flongle flow-cell. The method showed promise and worked as intended regarding the staphylococcal protein A-typing and the panel of resistance and virulence markers. However, the Multi-Locus Sequence Typing did still require optimization in order to be used clinically. In summary the project can be viewed as a success since it succeeded in being more time, cost and work efficient than many of its predecessors, when the problems with the Multi-Locus Sequence Typing are solved.

BackgroundMitochondrial genome sequences have become critical to the study of biodiversity. Genome skimming and other short-read based methods are the most common approaches, but they are not well-suited to scale up to multiplexing hundreds of samples. Here, we report on a new approach to sequence hundreds to thousands of complete mitochondrial genomes in parallel using long-amplicon sequencing. We amplified the mitochondrial genome of 677 specimens in two partially overlapping amplicons and implemented an asymmetric PCR-based indexing approach to multiplex 1,159 long amplicons together on a single PacBio SMRT Sequel II cell. We also tested this method on Oxford Nanopore Technologies (ONT) MinION R9.4 to assess if this method could be applied to other long-read technologies. We implemented several optimizations that make this method significantly more efficient than alternative mitochondrial genome sequencing methods.ResultsWith the PacBio sequencing data we recovered at least one of the two fragments for 96% of samples (~ 80-90%) with mean coverage ~ 1,500x. The ONT data recovered less than 50% of input fragments likely due to low throughput and the design of the Barcoded Universal Primers which were optimized for PacBio sequencing. We compared a single mitochondrial gene alignment to half and full mitochondrial genomes and found, as expected, increased tree support with longer alignments, though whole mitochondrial genomes were not significantly better than half mitochondrial genomes.ConclusionsThis method can effectively capture thousands of long amplicons in a single run and be used to build more robust phylogenies quickly and effectively. We provide several recommendations for future users depending on the evolutionary scale of their system. A natural extension of this method is to collect multi-locus datasets consisting of mitochondrial genomes and several long nuclear loci at once.

Methicillin-resistant Staphylococcus aureus (MRSA) is a variant of the more common Methicillin-Susceptible Staphylococcus aureus (MSSA), an opportunistic pathogen a portion of the human population carries as normal bacterial flora. When an outbreak of MRSA occurs, it is often important to determine if and how these strains are related to each other. In this report two different types of epidemiological methods were combined (namely Multi-Locus Sequence Typing and staphylococcal protein A-typing), in order to reduce workload, costs and time. A panel of resistance and virulence markers was also added to gather as much information about the culture as possible in a single analysis. To test the viability of the method extracted DNA and heat-treated bacterial cultures of both MRSA and MSSA were amplified with a curated panel of primers. These products were later sequenced with Nanopore’s MinION using the Flongle flow-cell. The method showed promise and worked as intended regarding the staphylococcal protein A-typing and the panel of resistance and virulence markers. However, the Multi-Locus Sequence Typing did still require optimization in order to be used clinically. In summary the project can be viewed as a success since it succeeded in being more time, cost and work efficient than many of its predecessors, when the problems with the Multi-Locus Sequence Typing are solved.

Detta arbete beskriver en undersökning som har utvärderat två navigeringstekniker: flockbeteenden och potential fields. I undersökningen har minioner i ett MOBA-scenario simulerat dessa två navigeringstekniker för att avgöra vilken av dem som ger den mjukaste navigeringen. Mjukhet i detta sammanhang innebär att minionerna kontinuerligt ska kunna navigera utan att fastna, samt minimera förändringar av riktningen mellan bilduppdateringar. En prototyp av ett MOBA-scenario utvecklades för att genomföra två experiment där mätningar togs när minionerna navigerade med de två navigeringsteknikerna. Resultatet visade att algoritmen för flockbeteendena medförde den mjukaste navigeringen. Dock konstaterades det att minionerna fastnade alltför ofta, vilket indikerar att framtida utveckling bör fokusera på att eliminera detta oönskade fenomen., Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.

We have adapted an established Ampliseq microhaplotype panel for nanopore sequencing with the Oxford Nanopore Technologies (ONT) system, as a cost-effective and highly scalable solution for forensic genetics applications. For this purpose, we designed a protocol combining direct PCR amplification from unextracted DNA with ONT library construction and sequencing using the MinION device and workflow. The analysis of reference samples at input amounts of 5–10 ng of DNA demonstrates stable coverage patterns, allele balance, and strand bias, reaching profile completeness and concordance rates of ∼95%. Similar levels were achieved when using direct-PCR from blood, buccal and semen swabs. Dilution series results indicate sensitivity is maintained down to 250 pg of input DNA, and informative profiles are produced down to 62.5 pg. Finally, we demonstrated the forensic utility of the nanopore workflow by analyzing two third degree pedigrees that showed low likelihood ratio values after the analysis of an extended panel of 38 STRs, achieving likelihood ratios 2–3 orders of magnitude higher when testing with the MinION–based haplotype data

Background: The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development. For instance, several strong, constitutive R. toruloides promoters have been characterized, but to date, only five inducible promoters have been identified. Although nitrogen-limited cultivation conditions are commonly used to induce lipid accumulation in this yeast, no promoters regulated by nitrogen starvation have been described for R. toruloides. Results: In this study, we used a combination of genomics and transcriptomics methods to identify novel R. toruloides promoter sequences that are either inducible or repressible by nitrogen starvation. RNA sequencing was used to assess gene expression in the recently isolated strain R. toruloides BOT-A2 during exponential growth and during nitrogen starvation, when cultivated with either glucose or xylose as the carbon source. The genome of BOT-A2 was sequenced using a combination of long- and short-read sequencing and annotated with support of the RNAseq data. Differential expression analysis was used to identify genes with a |log2 fold change|≥ 2 when comparing their expression during nitrogen depletion to that during exponential growth. The promoter regions from 16 of these genes were evaluated for their ability to drive the expression of a fluorescent reporter gene. Three promoters that were clearly upregulated under nitrogen starvation and three that were downregulated were selected and further characterized. One promoter, derived from gene RTBOTA2_003877, was found to function like an on–off switc

Background Oxford nanopore Technologies (ONT) provides three main library preparation strategies to sequence bacterial genomes. These include tagmentation (TAG), ligation (LIG) and amplification (PCR). Despite ONT’s recommendations, making an informed decision for preparation choice remains difficult without a side-by-side comparison. Here, we sequenced 12 bacterial strains to examine the overall output of these strategies, including sequencing noise, barcoding efficiency and assembly quality based on mapping to curated genomes established herein. Results Average read length ranged closely for TAG and LIG (> 5,000 bp), while being drastically smaller for PCR (< 1,100 bp). LIG produced the largest output with 33.62 Gbp vs. 11.72 Gbp for TAG and 4.79 Gbp for PCR. PCR produced the most sequencing noise with only 22.7% of reads mappable to the curated genomes, vs. 92.9% for LIG and 87.3% for TAG. Output per channel was most homogenous in LIG and most variable in PCR, while intermediate in TAG. Artifactual tandem content was most abundant in PCR (22.5%) and least in LIG and TAG (0.9% and 2.2%). Basecalling and demultiplexing of barcoded libraries resulted in ~ 20% data loss as unclassified reads and 1.5% read leakage. Conclusion The output of LIG was best (low noise, high read numbers of long lengths), intermediate in TAG (some noise, moderate read numbers of long lengths) and less desirable in PCR (high noise, high read numbers of short lengths). Overall, users should not accept assembly results at face value without careful replicon verification, including the detection of plasmids assembled from leaked reads.

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

Detta arbete beskriver en undersökning som har utvärderat två navigeringstekniker: flockbeteenden och potential fields. I undersökningen har minioner i ett MOBA-scenario simulerat dessa två navigeringstekniker för att avgöra vilken av dem som ger den mjukaste navigeringen. Mjukhet i detta sammanhang innebär att minionerna kontinuerligt ska kunna navigera utan att fastna, samt minimera förändringar av riktningen mellan bilduppdateringar. En prototyp av ett MOBA-scenario utvecklades för att genomföra två experiment där mätningar togs när minionerna navigerade med de två navigeringsteknikerna. Resultatet visade att algoritmen för flockbeteendena medförde den mjukaste navigeringen. Dock konstaterades det att minionerna fastnade alltför ofta, vilket indikerar att framtida utveckling bör fokusera på att eliminera detta oönskade fenomen., Det finns övrigt digitalt material (t.ex. film-, bild- eller ljudfiler) eller modeller/artefakter tillhörande examensarbetet som ska skickas till arkivet.There are other digital material (eg film, image or audio files) or models/artifacts that belongs to the thesis and need to be archived.

There are several affordable methods involving different sequencing technologies for microbial characterization. However, the choice of the sequencing platform and the downstream analysis can yield somewhat different results. Here we aimed to examine the strengths and limitations of different sequencing platforms for their use in aquacultured fish gut mucosal samples, using gilthead sea bream (Sparus aurata) as a model. The attention was focused on the portable Oxford Nanopore Technologies (ONT) MinION device, which offers the possibility of fast profiling of mucosal microbial samples. The standard PCR protocol provided for the MinION full-length sequencing of the 16S rRNA was optimized (input material, annealing temperature and number of cycles) for fish mucosal samples and the optimized protocol was validated using a standard mock community with known bacterial taxa and abundances. The optimized PCR (annealing temperature 52 °C, 30 cycles) yielded highly accurate results when sequencing mock community samples (R2 = 0.95) and was chosen for the subsequent analyses. Finally, the sequencing results were compared to those from Illumina MiSeq sequencing of the V3-V4 region of the 16S rRNA gene to determine strengths and weaknesses from both platforms. Our results showed that MinION is a reliable and accurate tool for the assessment of intestinal bacteria communities, yielding similar results to Illumina with correlation coefficients >0.75. However, biologically important but less abundant taxa (e.g. Actinobacteriota and Bacteroidota) were apparently masked when comparisons were made with the Illumina MiSeq results. We conclude that the choice of sequencing platform depends on the type of sample and scientific question. Thus, when evaluating fish gut mucosal samples, where the biological interpretation focuses on taxa related to gut function and metabolism, Illumina MiSeq allows a broader amplification of taxa of interest, while MinION provides good results in terms of

The Penicillia are known to produce a wide range natural products—some with devastating outcome for the agricultural industry and others with unexploited potential in different applications. However, a large-scale overview of the biosynthetic potential of different species has been lacking. In this study, we sequenced 93 Penicillium isolates and, together with eleven published genomes that hold similar assembly characteristics, we established a species phylogeny as well as defining a Penicillium pangenome. A total of 5612 genes were shared between ≥ 98 isolates corresponding to approximately half of the average number of genes a Penicillium genome holds. We further identified 15 lateral gene transfer events that have occurred in this collection of Penicillium isolates, which might have played an important role, such as niche adaption, in the evolution of these fungi. The comprehensive characterization of the genomic diversity in the Penicillium genus supersedes single-reference genomes, which do not necessarily capture the entire genetic variation.

Shortly after the beginning of the SARS-CoV-2 pandemic, many countries implemented sewage sentinel systems to monitor the circulation of the virus in the population. A fundamental part of these surveillance programs is the variant tracking through sequencing approaches to monitor and identify new variants or mutations that may be of importance. Two of the main sequencing platforms are Illumina and Oxford Nanopore Technologies. Here, we compare the performance of MiSeq (Illumina) and MinION (Oxford Nanopore Technologies), as well as two different data processing pipelines, to determine the effect they may have on the results. MiSeq showed higher sequencing coverage, lower error rate, and better capacity to detect and accurately estimate variant abundances than MinION R9.4.1 flow cell data. The use of different variant callers (LoFreq and iVar) and approaches to calculate the variant proportions had a remarkable impact on the results generated from wastewater samples. Freyja, coupled with iVar, may be more sensitive and accurate than LoFreq, especially with MinION data, but it comes at the cost of having a higher error rate. The analysis of MinION R10.4.1 flow cell data using Freyja combined with iVar narrows the gap with MiSeq performance in terms of read quality, accuracy, sensitivity, and number of detected mutations. Although MiSeq should still be considered as the standard method for SARS-CoV-2 variant tracking, MinION’s versatility and rapid turnaround time may represent a clear advantage during the ongoing pandemic.

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.

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.

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.

Environmental DNA (eDNA) comprises intra- and extra-cellular DNA molecules that are released in the environment and can be traced back to the source organism by analysing an environmental sample, such as water or sediment. While many limitations still exist, the coupling and integration of eDNA sampling with both traditional methods (direct capture) and/or other non-invasive approaches (imaging and acoustic data) has great potential to enhance monitoring capabilities of the marine environment. In addition, emerging technologies (including portable sequencers) are enabling the development of protocols for near real-time in situ applications of eDNA analysis. Our aim is to develop, test and implement eDNA metabarcoding protocols capable of being installed onto marine platforms such as marine fixed or mobile infrastructures (e.g. fixed observatories, platforms, research vessels) to provide real-time data of support to fisheries and other megafauna monitoring programs. An initial protocol has been already tested in laboratory conditions using a MinION portable sequencer (Oxford Nanopore Technologies), with preliminary results showing promising results in generating fish community profiles from eDNA samples collected at depth along the Northeast Atlantic continental shelf. Furter protocol development is ongoing and will include complete processing of environmental samples, from collection to High Throughput Sequencing (HTS) and bioinformatic analysis, in remote conditions. Specifically, results of protocol validation and implementation will be presented after execution in three contexts: (i) an aquarium with known species richness (Galway Atlantaquaria, Ireland); (ii) an open water fixed observatory equipped with a full suite of sensors including video (the Acqua Alta platform (Northern Adriatic Sea, Italy)); and (iii) offshore on board a research vessel during acoustic fisheries surveys (the Marine Institute's RV Celtic Explorer, Ireland). The inclusion of eDNA in the m

Fecal microbiota transplantation (FMT) is currently used for treating Clostridium difficile infection and explored for other clinical applications in experimental trials. However, the effectiveness of this therapy could vary, and partly depend on the donor's bacterial species engraftment, whose evaluation is challenging because there are no cost-effective strategies for accurately tracking the microbe transference. In this regard, the precise identification of bacterial species inhabiting the human gut is essential to define their role in human health unambiguously. We used Nanopore-based device to sequence bacterial rrn operons (16S-ITS-23S) and to reveal species-level abundance changes in the human gut microbiota of a FMT trial. By assessing the donor and recipient microbiota before and after FMT, we further evaluated whether this molecular approach reveals strain-level genetic variation to demonstrate microbe transfer and engraftment. Strict control over sequencing data quality and major microbiota covariates was critical for accurately estimating the changes in gut microbial species abundance in the recipients after FMT. We detected strain-level variation via single-nucleotide variants (SNVs) at rrn regions in a species-specific manner. We showed that it was possible to explore successfully the donor-bacterial strain (e.g., Parabacteroides merdae) engraftment in recipients of the FMT by assessing the nucleotide frequencies at rrn-associated SNVs. Our findings indicate that the engraftment of donors' microbiota is to some extent correlated with the improvement of metabolic health in recipients and that parameters such as the baseline gut microbiota configuration, sex, and age of donors should be considered to ensure the success of FMT in humans. The study was prospectively registered at the Dutch Trial registry - NTR4488 (https://www.trialregister.nl/trial/4488).

[Objective] To disseminate the portable sequencer MinION in developing countries for the main purpose of battling infectious diseases, we found a consortium called Global Research Alliance in Infectious Diseases (GRAID). By holding and inviting researchers both from developed and developing countries, we aim to train the participants with MinION’s operations and foster a collaboration in infectious diseases researches. As a real-life example in which resources are limited, we describe here a result from a training course, a metagenomics analysis from two blood samples collected from a routine cattle surveillance in Kulan Progo District, Yogyakarta Province, Indonesia in 2019., [Results] One of the samples was successfully sequenced with enough sequencing yield for further analysis. After depleting the reads mapped to host DNA, the remaining reads were shown to map to Theileria orientalis using BLAST and OneCodex. Although the reads were also mapped to Clostridium botulinum, those were found to be artifacts derived from the cow genome. An effort to construct a consensus sequence was successful using a reference-based approach with Pomoxis. Hence, we concluded that the asymptomatic cow might be infected with T. orientalis and showed the usefulness of sequencing technology, specifically the MinION platform, in a developing country.

The next generation sequencing technologies have revolutionised the generation of raw sequencedataof the human genome. Sequencing of human genomecan now be completed in a short span of time using the latest next-generation sequencing technology.Data of the size of terabytes and more is generated during the sequencing operation in real time.The complexity of data manipulation and management is a huge challenge facing the researcherstoday. The review provides an insight into the available nextgeneration sequencing technologies

The next generation sequencing technologies have revolutionised the generation of raw sequencedataof the human genome. Sequencing of human genomecan now be completed in a short span of time using the latest next-generation sequencing technology.Data of the size of terabytes and more is generated during the sequencing operation in real time.The complexity of data manipulation and management is a huge challenge facing the researcherstoday. The review provides an insight into the available nextgeneration sequencing technologies

The next generation sequencing technologies have revolutionised the generation of raw sequencedataof the human genome. Sequencing of human genomecan now be completed in a short span of time using the latest next-generation sequencing technology.Data of the size of terabytes and more is generated during the sequencing operation in real time.The complexity of data manipulation and management is a huge challenge facing the researcherstoday. The review provides an insight into the available nextgeneration sequencing technologies

The next generation sequencing technologies have revolutionised the generation of raw sequencedataof the human genome. Sequencing of human genomecan now be completed in a short span of time using the latest next-generation sequencing technology.Data of the size of terabytes and more is generated during the sequencing operation in real time.The complexity of data manipulation and management is a huge challenge facing the researcherstoday. The review provides an insight into the available nextgeneration sequencing technologies

The next generation sequencing technologies have revolutionised the generation of raw sequencedataof the human genome. Sequencing of human genomecan now be completed in a short span of time using the latest next-generation sequencing technology.Data of the size of terabytes and more is generated during the sequencing operation in real time.The complexity of data manipulation and management is a huge challenge facing the researcherstoday. The review provides an insight into the available nextgeneration sequencing technologies

The next generation sequencing technologies have revolutionised the generation of raw sequencedataof the human genome. Sequencing of human genomecan now be completed in a short span of time using the latest next-generation sequencing technology.Data of the size of terabytes and more is generated during the sequencing operation in real time.The complexity of data manipulation and management is a huge challenge facing the researcherstoday. The review provides an insight into the available nextgeneration sequencing technologies

In recent decades the conservation of cultural heritage has been attracting increasing research interest from many different scientific disciplines. Lately, the co-integration of chemistry and physics with biological techniques has shaped our understanding of the living microbial communities on sites of cultural heritage

In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking introduction, spread and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using established visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable "how-to" reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and long-read nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.

In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking introduction, spread and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using established visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable "how-to" reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and long-read nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.

In recent decades the conservation of cultural heritage has been attracting increasing research interest from many different scientific disciplines. Lately, the co-integration of chemistry and physics with biological techniques has shaped our understanding of the living microbial communities on sites of cultural heritage

In recent decades the conservation of cultural heritage has been attracting increasing research interest from many different scientific disciplines. Lately, the co-integration of chemistry and physics with biological techniques has shaped our understanding of the living microbial communities on sites of cultural heritage

The next generation sequencing technologies have revolutionised the generation of raw sequencedataof the human genome. Sequencing of human genomecan now be completed in a short span of time using the latest next-generation sequencing technology.Data of the size of terabytes and more is generated during the sequencing operation in real time.The complexity of data manipulation and management is a huge challenge facing the researcherstoday. The review provides an insight into the available nextgeneration sequencing technologies

We report whole-genome sequencing of influenza A virus (IAV) with 100% diagnostic sensitivity and results available in <24-48 h using amplicon-based nanopore sequencing technology (MinION) on clinical material from wild waterfowl (n = 19), commercial poultry (n = 4), and swine (n = 3). All 8 gene segments of IAV including those from 14 of the 18 recognized hemagglutinin subtypes and 9 of the 11 neuraminidase subtypes were amplified in their entirety at >500× coverage from each of 16 reference virus isolates evaluated. Subgenomic viral sequences obtained in 3 cases using Sanger sequencing as the reference standard were identical to those obtained when sequenced using the MinION approach. An inter-laboratory comparison demonstrated reproducibility when comparing 2 independent laboratories at ≥99.8% across the entirety of the IAV genomes sequenced.

Molecular information is crucial for species identification when facing challenging morphology-based specimen identifications. The use of DNA barcodes partially solves this problem, but in some cases when PCR is not an option (i.e., primers are not available, problems in reaction standardization), amplification-free approaches could be an optimal alternative. Recent advances in DNA sequencing, like the MinION device from Oxford Nanopore Technologies (ONT), allow to obtain genomic data with low laboratory and technical requirements, and at a relatively low cost. In this study, we explore ONT sequencing for molecular species identification from a total DNA sample obtained from a neotropical rodent and we also test the technology for complete mitochondrial genome reconstruction via genome skimming. We were able to obtain “de novo” the complete mitogenome of a specimen from the genus Melanomys (Cricetidae: Sigmodontinae) with average depth coverage of 78X using ONT-only data and by combining multiple assembly routines. Our pipeline for an automated species identification was able to identify the sample using unassembled sequence data (raw) in a reasonable computing time, which was substantially reduced when a priori information related to the organism identity was known. Our findings suggest ONT sequencing as a suitable candidate to solve species identification problems in metazoan nonmodel organisms and generate complete mtDNA datasets. © 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.

In recent decades the conservation of cultural heritage has been attracting increasing research interest from many different scientific disciplines. Lately, the co-integration of chemistry and physics with biological techniques has shaped our understanding of the living microbial communities on sites of cultural heritage

In recent decades the conservation of cultural heritage has been attracting increasing research interest from many different scientific disciplines. Lately, the co-integration of chemistry and physics with biological techniques has shaped our understanding of the living microbial communities on sites of cultural heritage

In recent decades the conservation of cultural heritage has been attracting increasing research interest from many different scientific disciplines. Lately, the co-integration of chemistry and physics with biological techniques has shaped our understanding of the living microbial communities on sites of cultural heritage

Background Rumen microorganisms carry antimicrobial resistance genes which pose a threaten to animals and humans in a One Health context. In order to tackle the emergence of antimicrobial resistance it is vital to understand how they appear, their relationship with the host, how they behave as a whole in the ruminal ecosystem or how they spread to the environment or humans. We sequenced ruminal samples from 416 Holstein dairy cows in 14 Spanish farms using nanopore technology, to uncover the presence of resistance genes and their potential effect on human, animal and environmental health. Results We found 998 antimicrobial resistance genes (ARGs) in the cow rumen and studied the 25 most prevalent genes in the 14 dairy cattle farms. The most abundant ARGs were related to the use of antibiotics to treat mastitis, metritis and lameness, the most common diseases in dairy cattle. The relative abundance (RA) of bacteriophages was positively correlated to the ARGs RA. The heritability of the RA of the more abundant ARGs ranged between 0.10 (mupA) and 0.49 (tetW), similar to the heritability of the RA of microbes that carried those ARGs. Even though these genes are carried by the microorganisms, the host is partially controlling their RA by having a more suitable rumen pH, folds, or other physiological traits that promote the growth of those microorganisms. Conclusions We were able to determine the most prevalent ARGs (macB, msbA, parY, rpoB2, tetQ and TaeA) in the ruminal bacteria ecosystem. The rumen is a reservoir of ARGs, and strategies to reduce the ARG load from livestock must be pursued.

Objectives: The assembly of fungal genomes using short-reads is challenged by long repetitive and low GC regions. However, long-read sequencing technologies, such as PacBio and Oxford Nanopore, are able to overcome many problematic regions, thereby providing an opportunity to improve fragmented genome assemblies derived from short reads only. Here, a necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) isolate 134 (Ptr134), which causes tan spot disease on wheat, was sequenced on a MinION using Oxford Nanopore Technologies (ONT), to improve on a previous Illumina short-read genome assembly and provide a more complete genome resource for pan-genomic analyses of Ptr. Results: The genome of Ptr134 sequenced on a MinION using ONT was assembled into 28 contiguous sequences with a total length of 40.79 Mb and GC content of 50.81%. The long-read assembly provided 6.79 Mb of new sequence and 2846 extra annotated protein coding genes as compared to the previous short-read assembly. This improved genome sequence represents near complete chromosomes, an important resource for large scale and pan genomic comparative analyses.

A rapid and accurate diagnostic assay represents an important means to detect Mycobacterium tuberculosis, identify drug-resistant strains and ensure treatment success. Currently employed techniques to diagnose drug-resistant tuberculosis include slow phenotypic tests or more rapid molecular assays that evaluate a limited range of drugs. Whole-genome-sequencing-based approaches can detect known drug-resistance-conferring mutations and novel variations; however, the dependence on growing samples in culture, and the associated delays in achieving results, represents a significant limitation. As an alternative, targeted sequencing strategies can be directly performed on clinical samples at high throughput. This study proposes a targeted sequencing assay to rapidly detect drug-resistant strains of M. tuberculosis using the Nanopore MinION sequencing platform. We designed a single-tube assay that targets nine genes associated with drug resistance to seven drugs and two phylogenetic-determining regions to determine strain lineage and tested it in nine clinical isolates and six sputa. The study's main aim is to calibrate MinNION variant calling to detect drug-resistance-associated mutations with different frequencies to match the accuracy of Illumina (the current gold-standard sequencing technology) from both culture and sputum samples. After calibrating Nanopore MinION variant calling, we demonstrated 100% agreement between Illumina WGS and our MinION set up to detect known drug resistance and phylogenetic variants in our dataset. Importantly, other variants in the amplicons are also detected, decreasing the recall. We identify minority variants and insertions/deletions as crucial bioinformatics challenges to fully reproduce Illumina WGS results.

[EN] Microorganisms can produce a wide variety of compounds and are key factors for understanding the behavior of ecological systems. Metagenomics is the tool for achieving this knowledge, studying microorganisms directly from their source through different approaches: focusing on sequencing marker genes (metataxonomics) or puzzling up the whole genetic material into separate genomes (metagenomics). In the recent past, Illumina has been the most sequencing technology used. However, short reads generated by Illumina are hard to assemble, and they produce very fragmented metagenomes. Despite their high intrinsic error, third generation sequencing platforms harbor the potential to overcome this issue thanks to their ability to generate longer reads. In this regard, MinION (Oxford Nanopore Technologies) is very advantageous for metagenomics applications, since it is cheap, portable and provides information in real-time. Many assemblers have been designed for dealing with error-prone data, but there is not a clear consensus about the tools to use to achieve the best results when handling MinION data. It is thus necessary to benchmark these tools and state what, why and when to use them. For that, using the best performing assemblers we know in the present, the aim of the present work is to analyze sequencing data from microbial communities of different complexities and systematically compare the metagenomes retrieved. The final goal of the study is to provide guidance for other scientists to choose the proper software, and to stimulate the rational development of tools and methodologies for this field., [ES] Los microorganismos producen compuestos muy variados y son esenciales para entender el comportamiento de sistemas ambientales. La metagenómica es la vía para obtener este conocimiento, estudiando los microorganismos directamente desde su hábitat usando diferentes enfoques: centrarse en secuenciar genes marcadores (metataxonómica), o montar todo el material genético en genomas separados (metagenómica). En los últimos años, Illumina ha sido la tecnología más usada para secuenciar; sin embargo, las lecturas cortas que genera Illumina son difíciles de ensamblar, y producen metagenomas muy fragmentados. A pesar de su alto error intrínseco, las plataformas de tercera generación tienen potencial para superar este inconveniente gracias a que pueden generar lecturas más largas. En este contexto, MinION (Oxford Nanopore Technologies) es muy conveniente para aplicaciones metagenómicas; ya que es barato, portable y proporciona información en tiempo real. Se han diseñado muchos ensambladores para tratar con datos de mayor error intrínseco, pero no hay un consenso en cuanto a las herramientas que se deben usar para obtener los mejores resultados cuando se usa MinION. Es por tanto necesario evaluar estas herramientas y definir qué, por qué y cuándo usarlas. Para este propósito, y usando los mejores ensambladores que se conocen actualmente, la finalidad de este proyecto es analizar datos secuenciados de comunidades microbianas de varias complejidades y comparar sistemáticamente los metagenomas obtenidos, con el objetivo de orientar a otros científicos para elegir el software adecuado y estimular el desarrollo racional de nuevas herramientas y estrategias para este campo.

[EN] Microorganisms can produce a wide variety of compounds and are key factors for understanding the behavior of ecological systems. Metagenomics is the tool for achieving this knowledge, studying microorganisms directly from their source through different approaches: focusing on sequencing marker genes (metataxonomics) or puzzling up the whole genetic material into separate genomes (metagenomics). In the recent past, Illumina has been the most sequencing technology used. However, short reads generated by Illumina are hard to assemble, and they produce very fragmented metagenomes. Despite their high intrinsic error, third generation sequencing platforms harbor the potential to overcome this issue thanks to their ability to generate longer reads. In this regard, MinION (Oxford Nanopore Technologies) is very advantageous for metagenomics applications, since it is cheap, portable and provides information in real-time. Many assemblers have been designed for dealing with error-prone data, but there is not a clear consensus about the tools to use to achieve the best results when handling MinION data. It is thus necessary to benchmark these tools and state what, why and when to use them. For that, using the best performing assemblers we know in the present, the aim of the present work is to analyze sequencing data from microbial communities of different complexities and systematically compare the metagenomes retrieved. The final goal of the study is to provide guidance for other scientists to choose the proper software, and to stimulate the rational development of tools and methodologies for this field., [ES] Los microorganismos producen compuestos muy variados y son esenciales para entender el comportamiento de sistemas ambientales. La metagenómica es la vía para obtener este conocimiento, estudiando los microorganismos directamente desde su hábitat usando diferentes enfoques: centrarse en secuenciar genes marcadores (metataxonómica), o montar todo el material genético en genomas separados (metagenómica). En los últimos años, Illumina ha sido la tecnología más usada para secuenciar; sin embargo, las lecturas cortas que genera Illumina son difíciles de ensamblar, y producen metagenomas muy fragmentados. A pesar de su alto error intrínseco, las plataformas de tercera generación tienen potencial para superar este inconveniente gracias a que pueden generar lecturas más largas. En este contexto, MinION (Oxford Nanopore Technologies) es muy conveniente para aplicaciones metagenómicas; ya que es barato, portable y proporciona información en tiempo real. Se han diseñado muchos ensambladores para tratar con datos de mayor error intrínseco, pero no hay un consenso en cuanto a las herramientas que se deben usar para obtener los mejores resultados cuando se usa MinION. Es por tanto necesario evaluar estas herramientas y definir qué, por qué y cuándo usarlas. Para este propósito, y usando los mejores ensambladores que se conocen actualmente, la finalidad de este proyecto es analizar datos secuenciados de comunidades microbianas de varias complejidades y comparar sistemáticamente los metagenomas obtenidos, con el objetivo de orientar a otros científicos para elegir el software adecuado y estimular el desarrollo racional de nuevas herramientas y estrategias para este campo.

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

Newcastle disease (ND) is one of the most challenging infectious diseases affecting poultry production in Africa, causing major economic losses. To date, Newcastle disease virus isolates from several African countries have been grouped into class II NDV genotypes I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII and XXI. Although ND is endemic in many African countries, information on circulating genotypes is still scarce. In Tanzania, outbreaks with genotypes V and XIII have been reported. In West and Central Africa, genotypes XIV, XVII, and XVIII are the most predominant. To investigate other genotypes circulating in Tanzania and Ghana, we performed molecular genotyping on isolates from Tanzania and Ghana using the MinION, a third-generation portable sequencing device from Oxford Nanopore Technologies. Using the MinION, we successfully sequenced the NDV F gene hypervariable region of 24 isolates from Tanzania and four samples from Ghana. In Tanzania, genotypes V, VII and XIII were detected. All isolates from Ghana belonged to genotype XVIII. The data obtained in this study reflect the genetic diversity of NDV in Africa and highlight the importance of surveillance for monitoring the distribution of NDV genotypes and viral evolution.

Newcastle disease (ND) is one of the most challenging infectious diseases affecting poultry production in Africa, causing major economic losses. To date, Newcastle disease virus isolates from several African countries have been grouped into class II NDV genotypes I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII and XXI. Although ND is endemic in many African countries, information on circulating genotypes is still scarce. In Tanzania, outbreaks with genotypes V and XIII have been reported. In West and Central Africa, genotypes XIV, XVII, and XVIII are the most predominant. To investigate other genotypes circulating in Tanzania and Ghana, we performed molecular genotyping on isolates from Tanzania and Ghana using the MinION, a third-generation portable sequencing device from Oxford Nanopore Technologies. Using the MinION, we successfully sequenced the NDV F gene hypervariable region of 24 isolates from Tanzania and four samples from Ghana. In Tanzania, genotypes V, VII and XIII were detected. All isolates from Ghana belonged to genotype XVIII. The data obtained in this study reflect the genetic diversity of NDV in Africa and highlight the importance of surveillance for monitoring the distribution of NDV genotypes and viral evolution.

[EN] Microorganisms can produce a wide variety of compounds and are key factors for understanding the behavior of ecological systems. Metagenomics is the tool for achieving this knowledge, studying microorganisms directly from their source through different approaches: focusing on sequencing marker genes (metataxonomics) or puzzling up the whole genetic material into separate genomes (metagenomics). In the recent past, Illumina has been the most sequencing technology used. However, short reads generated by Illumina are hard to assemble, and they produce very fragmented metagenomes. Despite their high intrinsic error, third generation sequencing platforms harbor the potential to overcome this issue thanks to their ability to generate longer reads. In this regard, MinION (Oxford Nanopore Technologies) is very advantageous for metagenomics applications, since it is cheap, portable and provides information in real-time. Many assemblers have been designed for dealing with error-prone data, but there is not a clear consensus about the tools to use to achieve the best results when handling MinION data. It is thus necessary to benchmark these tools and state what, why and when to use them. For that, using the best performing assemblers we know in the present, the aim of the present work is to analyze sequencing data from microbial communities of different complexities and systematically compare the metagenomes retrieved. The final goal of the study is to provide guidance for other scientists to choose the proper software, and to stimulate the rational development of tools and methodologies for this field., [ES] Los microorganismos producen compuestos muy variados y son esenciales para entender el comportamiento de sistemas ambientales. La metagenómica es la vía para obtener este conocimiento, estudiando los microorganismos directamente desde su hábitat usando diferentes enfoques: centrarse en secuenciar genes marcadores (metataxonómica), o montar todo el material genético en genomas separados (metagenómica). En los últimos años, Illumina ha sido la tecnología más usada para secuenciar; sin embargo, las lecturas cortas que genera Illumina son difíciles de ensamblar, y producen metagenomas muy fragmentados. A pesar de su alto error intrínseco, las plataformas de tercera generación tienen potencial para superar este inconveniente gracias a que pueden generar lecturas más largas. En este contexto, MinION (Oxford Nanopore Technologies) es muy conveniente para aplicaciones metagenómicas; ya que es barato, portable y proporciona información en tiempo real. Se han diseñado muchos ensambladores para tratar con datos de mayor error intrínseco, pero no hay un consenso en cuanto a las herramientas que se deben usar para obtener los mejores resultados cuando se usa MinION. Es por tanto necesario evaluar estas herramientas y definir qué, por qué y cuándo usarlas. Para este propósito, y usando los mejores ensambladores que se conocen actualmente, la finalidad de este proyecto es analizar datos secuenciados de comunidades microbianas de varias complejidades y comparar sistemáticamente los metagenomas obtenidos, con el objetivo de orientar a otros científicos para elegir el software adecuado y estimular el desarrollo racional de nuevas herramientas y estrategias para este campo.

Fisheries enforcement relies on visual catch identification and quantification at sea or when landed. Silage (fish dissolved in acid) and fish blocks (block frozen fish) are promising methods for on-board processing and storage of low-value catches. We examined the use of nondestructive sampling and two DNA-based methods, quantitative PCR (qPCR) and metabarcoding, to assess species composition and relative abundance in industrial grade experimental silage and fish blocks. We demonstrate the ability to identify and quantify DNA from fish species in both products. qPCR analysis of small silage samples collected over 21 days detected all target control species. DNA from one species (Atlantic wolffish) was consistently overrepresented while, for three species of gadoids (Atlantic cod, haddock and whiting), the DNA content matched input tissue proportions with high accuracy. qPCR and metabarcoding of fish blocks, sampled as run-off water and exterior swabs, provided consistent species detection, with the highest variance observed in quantification from swab samples. Our analysis shows that DNA-based methods have significant potential as a tool for species identification and quantification of complex on-board-processed seafood products and are readily applicable to taxonomically and morphologically similar fish. There is, however, a need for establishing DNA/weight calibration factors for primary fisheries species.

Infectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.

Background: Riverine ecosystems are biogeochemical powerhouses driven largely by microbial communities that inhabit water columns and sediments. Because rivers are used extensively for anthropogenic purposes (drinking water, recreation, agriculture, and industry), it is essential to understand how these activities affect the composition of river microbial consortia. Recent studies have shown that river metagenomes vary considerably, suggesting that microbial community data should be included in broad-scale river ecosystem models. But such ecogenomic studies have not been applied on a broad "aquascape"scale, and few if any have applied the newest nanopore technology. Results: We investigated the metagenomes of 11 rivers across 3 continents using MinION nanopore sequencing, a portable platform that could be useful for future global river monitoring. Up to 10 Gb of data per run were generated with average read lengths of 3.4 kb. Diversity and diagnosis of river function potential was accomplished with 0.5-1.0 ⋅ 106 long reads. Our observations for 7 of the 11 rivers conformed to other river-omic findings, and we exposed previously unrecognized microbial biodiversity in the other 4 rivers. Conclusions: Deeper understanding that emerged is that river microbial consortia and the ecological functions they fulfil did not align with geographic location but instead implicated ecological responses of microbes to urban and other anthropogenic effects, and that changes in taxa manifested over a very short geographic space.

Fisheries enforcement relies on visual catch identification and quantification at sea or when landed. Silage (fish dissolved in acid) and fish blocks (block frozen fish) are promising methods for on-board processing and storage of low-value catches. We examined the use of nondestructive sampling and two DNA-based methods, quantitative PCR (qPCR) and metabarcoding, to assess species composition and relative abundance in industrial grade experimental silage and fish blocks. We demonstrate the ability to identify and quantify DNA from fish species in both products. qPCR analysis of small silage samples collected over 21 days detected all target control species. DNA from one species (Atlantic wolffish) was consistently overrepresented while, for three species of gadoids (Atlantic cod, haddock and whiting), the DNA content matched input tissue proportions with high accuracy. qPCR and metabarcoding of fish blocks, sampled as run-off water and exterior swabs, provided consistent species detection, with the highest variance observed in quantification from swab samples. Our analysis shows that DNA-based methods have significant potential as a tool for species identification and quantification of complex on-board-processed seafood products and are readily applicable to taxonomically and morphologically similar fish. There is, however, a need for establishing DNA/weight calibration factors for primary fisheries species.