Freshwater monitoring by nanopore sequencing

Urban, Lara; Holzer, Andre; Baronas, J. Jotautas; Hall, Michael B.; Braeuninger‐Weimer, Philipp; Scherm, Michael J.; Kunz, Daniel J.; Perera, Surangi; Martin-Herranz, Daniel E.; Tipper, Edward T.; Salter, Susannah J.; Stammnitz, Maximilian R

doi:10.7554/elife.61504

Cited by 86 publications

(90 citation statements)

References 88 publications

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“…Due in part to these key advantages-ease of library preparation, the small and portable footprint of ONT sequencing instruments, and the generation of real-time sequencing datananopore sequencing is particularly well-suited for the purposes of pathogen detection and biosurveillance [14,[16][17][18]. In contrast to conventional surveillance methods, which rely heavily on nucleic acid amplification (i.e., PCR), pathogen detection by nanopore sequencing can: (1) bypass the requirement that investigators possess a priori knowledge of the pathogen(s) being targeted, (2) enable detection of many agents simultaneously, and (3) provide a greater scope of downstream genomic sequence information suitable for pathogen strain typing, functional characterization, and phylogenetics [16,19].…”

Section: Introductionmentioning

confidence: 99%

Enabling metagenomic surveillance for bacterial tick-borne pathogens using nanopore sequencing with adaptive sampling

et al. 2021

Preprint

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Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and surveillance. In particular, single-molecule nucleotide sequence data originating from Oxford Nanopore Technologies (ONT) sequencing platforms can be bioinformatically leveraged, in real-time, for enhanced biosurveillance of a vast array of zoonoses. The recently released nanopore adaptive sampling (NAS) pipeline facilitates immediate mapping of individual nucleotide molecules (i.e., DNA, cDNA, and RNA) to a given reference as each molecule is sequenced. User-defined thresholds then allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. Here, we show how NAS can be used to selectively sequence entire genomes of bacterial tick-borne pathogens circulating in wild populations of the blacklegged tick vector, Ixodes scapularis. The NAS method provided a two-fold increase in targeted pathogen sequences, successfully enriching for Borrelia (Borreliella) burgdorferi s.s.; Borrelia (Borrelia) miyamotoi; Anaplasma phagocytophilum; and Ehrlichia muris eauclairensis genomic DNA within our I. scapularis samples. Our results indicate that NAS has strong potential for real-time sequence-based pathogen surveillance.

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Section: Introductionmentioning

confidence: 99%

Enabling metagenomic surveillance for bacterial tick-borne pathogens using nanopore sequencing with adaptive sampling

et al. 2021

Preprint

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“…16s rDNA sequencing is based on partial similarity in the complete gene with an approximate length of 1540 bases and variety in nine hypervariable regions unique for each bacterial genus [ 127 ]. Unfortunately, the first and second generation of sequencing depend on a relatively large and stationary apparatus with high running costs [ 128 ]. This changed after the introduction of the third generation of sequencers developed by Oxford Nanopore Technology.…”

Section: Detection Of Sars-cov-2mentioning

confidence: 99%

Wastewater-Based Epidemiology as an Early Warning System for the Spreading of SARS-CoV-2 and Its Mutations in the Population

Martı́n

Gál

Špalková

et al. 2021

IJERPH

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New methodologies based on the principle of “sewage epidemiology” have been successfully applied before in the detection of illegal drugs. The study describes the idea of early detection of a virus, e.g., SARS-CoV-2, in wastewater in order to focus on the area of virus occurrence and supplement the results obtained from clinical examination. By monitoring temporal variation in viral loads in wastewater in combination with other analysis, a virus outbreak can be detected and its spread can be suppressed early. The use of biosensors for virus detection also seems to be an interesting application. Biosensors are highly sensitive, selective, and portable and offer a way for fast analysis. This manuscript provides an overview of the current situation in the area of wastewater analysis, including genetic sequencing regarding viral detection and the technological solution of an early warning system for wastewater monitoring based on biosensors.

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“…Another study evaluating long-read sequencing of the 16S rRNA gene (amplicon sequencing) in water from informal settlements in Nepal found that amplicon sequencing correlated with qPCR results for detection of Vibrio cholera but recommended combined use to avoid false positives 24 . Other studies have found nanopore sequencing (metagenomic and amplicon) useful for profiling spatial and temporal variation in the core communities and function of bacteria in river samples 53,54 .…”

Section: Utility Of Assembled Long Readsmentioning

confidence: 99%

Surveillance of potential pathogens and antibiotic resistance in wastewater and surface water from Boston, USA and Vellore, India using long-read metagenomic sequencing

Voth-Gaeddert

Metilda

et al. 2021

Preprint

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Environmental sampling (wastewater) could be an efficient surveillance strategy to capture global emerging trends in the spread of antibiotic resistance. Long-read DNA sequencing can resolve the genetic context of antibiotic resistance genes (ARGs) and is a promising tool for non-culture-based monitoring of antibiotic-resistant pathogens and ARGs in environmental samples, but has not been rigorously validated against conventional methods. We tested long-read sequencing using the portable Nanopore MinION for surveying pathogens, ARGs, and antibiotic-resistant pathogens in municipal wastewater, hospital wastewater, and surface water collected from Boston, USA and Vellore, India. We compared detection of enteric pathogens by assembly of long reads, with and without short-read polishing, and unassembled raw long reads for ARGs to multiplex real-time PCR. Using real-time PCR as a benchmark, long-read metagenomics was 49% sensitive and 75% specific at pathogen detection in assembled contigs, and 16% sensitive and 100% specific at detecting 28 clinically relevant resistance genes in raw long reads. Short-read polishing did not substantially improve pathogen identification or impact ARG identification in the assembled contigs, demonstrating that short-read polishing is not required, which greatly reduces costs. The high specificity of ARG detection supports portable long-read sequencing as a valuable tool to profile ARGs and antibiotic-resistant pathogens for environmental surveillance programs.

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Freshwater monitoring by nanopore sequencing

Cited by 86 publications

References 88 publications

Enabling metagenomic surveillance for bacterial tick-borne pathogens using nanopore sequencing with adaptive sampling

Enabling metagenomic surveillance for bacterial tick-borne pathogens using nanopore sequencing with adaptive sampling

Wastewater-Based Epidemiology as an Early Warning System for the Spreading of SARS-CoV-2 and Its Mutations in the Population

Surveillance of potential pathogens and antibiotic resistance in wastewater and surface water from Boston, USA and Vellore, India using long-read metagenomic sequencing

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