Unbiased Strain-Typing of Arbovirus Directly from Mosquitoes Using Nanopore Sequencing: A Field-forward Biosurveillance Protocol

Russell, Joseph A.; Campos, Brittany; Stone, Jennifer; Blosser, Erik M.; Burkett‐Cadena, Nathan D.; Jacobs, Jonathan L.

doi:10.1101/183780

Cited by 5 publications

(5 citation statements)

References 64 publications

(60 reference statements)

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“…The area under the curve (AUC) is calculated using the 'step' method. Most assays achieve a 100% sensitivity and 0% FPR near a read count cutoff of 10 for the highest 3 concentrations (3, 4, and 5), and many achieve this AUC at the lowest 2 (1,2), with the exception of PRC_30 Fig. 11 Real-time PCR PR and ROC curves for assays having a real time PCR false negative or false positive.…”

Section: Discussionmentioning

confidence: 99%

“…Nucleic acid sequencing-based bioagent detection applications have recently gained momentum in the microbial diagnostics and biosurveillance arenas [1,2]. Historically, however, the field of human genetics has led the way in advancing sequence-based diagnostics, following the advent of Next Generation Sequencing (NGS) technologies just over a decade ago [3].…”

Section: Introductionmentioning

confidence: 99%

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Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens

et al. 2020

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Background: The state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for highthroughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments. Results: To address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5-50 Colony Forming Units (CFU)/ml).

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens

et al. 2020

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“…The metagenomic amplification and sequencing technologies used here are fairly portable and can be adapted to complete these analyses at sea within a 24‐hour window. Similar field‐based applications have been applied for the rapid detection of infectious pathogens (Russell et al 2018) and the methods presented in our paper would allow for real‐time health assessments of cetacean populations in future sampling expeditions, as well as the initiation of more sophisticated experimental designs. These are all examples of types of information that can be extracted from blow and contribute to our knowledge of the health or well‐being of large whales.…”

Section: Management Implicationsmentioning

confidence: 90%

Genetic, Endocrine, and Microbiological Assessments of Blue, Humpback and Killer Whale Health using Unoccupied Aerial Systems

Atkinson

Rogan²,

Baker

et al. 2021

Wildlife Society Bulletin

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Unoccupied aerial system (UAS) technologies applied to health assessments of large whales can have positive implications for progressive management. We focused on the collection of cetacean respiratory blow samples for endocrine, DNA profiling, microbial metabarcoding, and metagenomics analyses, with the goal of improving management of large whale populations. Blow samples were collected from humpback (Megaptera novaeangliae, n = 109 samples analyzed), blue (Balaenoptera musculus, n = 21 samples analyzed), and killer whale (Orcinus orca, n = 1 sample analyzed) species, as well as the responses of the whales to the collection of their blow by UAS. Endocrine analyses were validated for 5 steroid hormones in humpback whales and 4 hormones in blue whales. For DNA profiling, we attempted to extract and amplify nuclear and mitochondrial DNA, resulting in sequencing of mtDNA haplotypes for 54% of samples, identification of sex for 39%, and individual identification by microsatellite genotyping for 17%. The DNA profiles of 2 of the blow samples from humpback whales were matched to a DNA register for this regional population. Metagenomic and microbial metabarcoding classifications found a diverse number of bacteria, eukaryotes, and viruses in humpback whale blow. Although a significant portion of classifications were found in both seawater and blow, several of the most abundant organisms were present only in blow samples, suggesting they are true members of the respiratory microbiome. A comprehensive integration of laboratory‐based approaches using noninvasive UAS collection technologies could become an important management tool for health assessments of large cetaceans, especially for species listed as endangered. The addition of individual and population‐level health assessments to currently practiced stewardship of large whales, renders them as excellent sentinels of ocean health. © 2021 The Wildlife Society.

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“…The MinION is a pocket-sized, USB-powered nanopore sequencing platform, weighing less than 100 g, yet capable of directly sequencing full-length DNA or RNA sequences (Russell et al, 2018;Urban et al, 2015). Direct RNA sequencing was performed using the Direct RNA Sequencing protocol with the SQK-RNA001 kit (ONT, USA, 2018a), with 700 ng of RNA (to ensure sufficient material remained for sequencing) used as input for library preparation.…”

Section: Nanopore Library Preparation and Direct Rna Sequencing By Ontmentioning

confidence: 99%

Evaluating the potential of direct RNA nanopore sequencing: Metatranscriptomics highlights possible seasonal differences in a marine pelagic crustacean zooplankton community

Semmouri

Schamphelaere

Mees

et al. 2020

Marine Environmental Research

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The implementation of cost-effective monitoring programs for zooplankton remains challenging due to the requirements of taxonomical expertise and the high costs of sampling and species identification. To reduce costs, molecular methods have been proposed as alternatives to morphology-based monitoring. Metatranscriptomics can contribute to promote both cost-effectiveness and accuracy of biological assessments of aquatic ecosystems. Here, we describe and evaluate the construction of a metatranscriptome dataset from a pelagic crustacean zooplankton community. We sampled zooplankton in one marine station, named LW02, in the North Sea, in both winter and summer, and generated transcripts using Oxford Nanopore Technology (ONT), a third-generation nanopore-based sequencing technology. ONT is, uniquely, capable of sequencing RNA directly, rather than depending on reverse transcription and PCR, and applicable to be used directly in the field. We found that metatranscriptomics is capable of species detection, including screening for the presence of endoparasites, hence competing with morphological identification. Taxonomic analysis based on ribosomal 18S transcripts identified calanoid copepods, particularly Temora longicornis and Acartia clausi, as the most abundant community members. Moreover, up to 40.4% and 50.5% of all sequences could be assigned to predicted genes in the winter and summer sample, respectively. The most abundant mRNA transcripts with known function coded for essential metabolic processes. GO term annotation revealed that genes involved in glycolytic and translation-related processes were most expressed in the community. Although small in scale, our study provides the basis for future efforts to characterize the metatranscriptome of marine zooplankton communities and its application in biomonitoring programs.

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Unbiased Strain-Typing of Arbovirus Directly from Mosquitoes Using Nanopore Sequencing: A Field-forward Biosurveillance Protocol

Abstract: 12The future of infectious disease surveillance and outbreak response is trending towards smaller 13 hand-held solutions for point-of-need pathogen detection.

Cited by 5 publications

References 64 publications

Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens

Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens

Genetic, Endocrine, and Microbiological Assessments of Blue, Humpback and Killer Whale Health using Unoccupied Aerial Systems

Evaluating the potential of direct RNA nanopore sequencing: Metatranscriptomics highlights possible seasonal differences in a marine pelagic crustacean zooplankton community

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