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:10.1101/2020.02.06.936302

Cited by 8 publications

(11 citation statements)

References 84 publications

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“…Using Minimap2 (Li, 2018), we aligned our nanopore sequencing reads against the three different publicly available databases, NCBI 16S RefSeq, RDP and SILVA. Performance matrix comparison of thirteen different classification tools by Urban and colleagues revealed that Minimap2 provided robust alignments that were closely aligned to their mock community taxa (Urban et al, 2020). However, similar to the challenges encountered by Urban et al (2020), we have had issues of high memory usage on Minimap2, which necessitated a reduction in the number bases loaded into memory to process in the query batch (command -K 100M).…”

Section: Discussionmentioning

confidence: 99%

“…Performance matrix comparison of thirteen different classification tools by Urban and colleagues revealed that Minimap2 provided robust alignments that were closely aligned to their mock community taxa (Urban et al, 2020). However, similar to the challenges encountered by Urban et al (2020), we have had issues of high memory usage on Minimap2, which necessitated a reduction in the number bases loaded into memory to process in the query batch (command -K 100M). By comparing against a defined mock community, we observed differences in the taxonomic assignments between the databases-with the NCBI 16S RefSeq database clustered at 100% providing the most accurate assignments, which could be attributed to the differences in the database size and sequence validation steps (Balvočiūtė & Huson, 2017;Park & Won, 2018).…”

Section: Discussionmentioning

confidence: 99%

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Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis

Low

Fuentes-Utrilla

Hodson

et al. 2021

PeerJ

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Background Microbial keratitis is a leading cause of preventable blindness worldwide. Conventional sampling and culture techniques are time-consuming, with over 40% of cases being culture-negative. Nanopore sequencing technology is portable and capable of generating long sequencing reads in real-time. The aim of this study is to evaluate the potential of nanopore sequencing directly from clinical samples for the diagnosis of bacterial microbial keratitis. Methods Using full-length 16S rRNA amplicon sequences from a defined mock microbial community, we evaluated and benchmarked our bioinformatics analysis pipeline for taxonomic assignment on three different 16S rRNA databases (NCBI 16S RefSeq, RDP and SILVA) with clustering at 97%, 99% and 100% similarities. Next, we optimised the sample collection using an ex vivo porcine model of microbial keratitis to compare DNA recovery rates of 12 different collection methods: 21-gauge needle, PTFE membrane (4 mm and 6 mm), Isohelix™ SK-2S, Sugi® Eyespear, Cotton, Rayon, Dryswab™, Hydraflock®, Albumin-coated, Purflock®, Purfoam and Polyester swabs. As a proof-of-concept study, we then used the sampling technique that provided the highest DNA recovery, along with the optimised bioinformatics pipeline, to prospectively collected samples from patients with suspected microbial keratitis. The resulting nanopore sequencing results were then compared to standard microbiology culture methods. Results We found that applying alignment filtering to nanopore sequencing reads and aligning to the NCBI 16S RefSeq database at 100% similarity provided the most accurate bacterial taxa assignment. DNA concentration recovery rates differed significantly between the collection methods (p < 0.001), with the Sugi® Eyespear swab providing the highest mean rank of DNA concentration. Then, applying the optimised collection method and bioinformatics pipeline directly to samples from two patients with suspected microbial keratitis, sequencing results from Patient A were in agreement with culture results, whilst Patient B, with negative culture results and previous antibiotic use, showed agreement between nanopore and Illumina Miseq sequencing results. Conclusion We have optimised collection methods and demonstrated a novel workflow for identification of bacterial microbial keratitis using full-length 16S nanopore sequencing.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis

Low

Fuentes-Utrilla

Hodson

et al. 2021

PeerJ

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“…The described data processing and read classification steps were implemented using the Snakemake workflow management system ( Köster and Rahmann, 2012 ) and are available on Github - together with all necessary downstream analysis scripts to reproduce the results of this manuscript ( https://github.com/d-j-k/puntseq ; Urban et al, 2020 ; copy archived at swh:1:rev:1408d508c807b88e0989a5252c5d904072dc3c4a ).…”

Section: Methodsmentioning

confidence: 99%

“…All classification assessment steps and summary statistics were performed in R or Python ( https://github.com/d-j-k/puntseq ; Urban et al, 2020 ). We used the Python package scikit-bio for the calculation of the Simpson index and the Shannon's diversity as well as equitability index.…”

Section: Methodsmentioning

confidence: 99%

Freshwater monitoring by nanopore sequencing

Urban

Holzer

Baronas

et al. 2021

eLife

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While traditional microbiological freshwater tests focus on the detection of specific bacterial indicator species, including pathogens, direct tracing of all aquatic DNA through metagenomics poses a profound alternative. Yet, in situ metagenomic water surveys face substantial challenges in cost and logistics. Here, we present a simple, fast, cost-effective and remotely accessible freshwater diagnostics workflow centred around the portable nanopore sequencing technology. Using defined compositions and spatiotemporal microbiota from surface water of an example river in Cambridge (UK), we provide optimised experimental and bioinformatics guidelines, including a benchmark with twelve taxonomic classification tools for nanopore sequences. We find that nanopore metagenomics can depict the hydrological core microbiome and fine temporal gradients in line with complementary physicochemical measurements. In a public health context, these data feature relevant sewage signals and pathogen maps at species level resolution. We anticipate that this framework will gather momentum for new environmental monitoring initiatives using portable devices.

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“…Recently, a work from Urban et al . [ 44 ] studied the microbial communities present in the surface water of Cam River (Cambridge). All the protocols were carried out in the lab, and the authors were able to achieve up to ∼5.5 M 16S rRNA full-length sequences with exclusive barcode assignments in a single MinION run.…”

Section: Portable Sequencing In Natural Environmentsmentioning

confidence: 99%

A lab in the field: applications of real-time, in situ metagenomic sequencing

Latorre‐Pérez¹,

Pascual²,

Porcar

et al. 2020

Biology Methods and Protocols

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High-throughput metagenomic sequencing is considered one of the main technologies fostering the development of microbial ecology. Widely-used second generation sequencers have enabled the analysis of extremely diverse microbial communities, the discovery of novel gene functions, and the comprehension of the metabolic interconnections established among microbial consortia. However, the high cost of the sequencers and the complexity of library preparation and sequencing protocols still hamper the application of metagenomic sequencing in a vast range of real-life applications. In this context, the emergence of portable, third-generation sequencers is becoming a popular alternative for the rapid analysis of microbial communities in particular scenarios, due to their low cost, simplicity of operation, and rapid yield of results. This review discusses the main applications of real-time, in situ metagenomic sequencing developed to date, highlighting the relevance of this technology in current challenges (such as the management of global pathogen outbreaks) and in the next future of industry and clinical diagnosis.

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Freshwater monitoring by nanopore sequencing

Cited by 8 publications

References 84 publications

Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis

Evaluation of full-length nanopore 16S sequencing for detection of pathogens in microbial keratitis

Freshwater monitoring by nanopore sequencing

A lab in the field: applications of real-time, in situ metagenomic sequencing

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