Blog: PuntSeq - Portable nanopore sequencing of freshwater metagenomes

In this blog, Lara Urban discusses the importance of freshwater microbial monitoring, and the founding of PuntSeq – a citizen science effort centred around using nanopore sequencing technology to gain a comprehensive picture of the microbes present in the River Cam, Cambridge.

The assurance of access to safe drinking water has been recognised as a United Nations Millennium Development Goal (Bartram et al., 2005). Global trends, such as climate change and agricultural intensification, reinforce this requirement, and increasingly require comprehensive freshwater monitoring frameworks. Ideally, these frameworks would combine cost effectiveness and technology which enables rapid analysis, with deployability in the field. While traditional microbial freshwater tests have focused on the detection of specific bacterial indicator organisms as well as pathogens, direct tracing of all aquatic DNA through metagenomics approaches poses a profound alternative. Such microbial metagenomics approaches allow detection of all bacterial organisms present in a sample through high-throughput DNA sequencing, and thus yield informative measures of the presence, but also the functional diversity, of an environment's microbiome (Tringe & Rubin, 2005).

PuntSeq: A citizen science project

In Cambridge, UK, river users like rowers and punters obtain infections associated with pathogens from the local river every year. As an information and research framework that targets the involved microbial culprits was lacking, we founded PuntSeq; this is as a citizen science effort that aims to provide an in-depth resolution of the River Cam pathogen landscape and, on a larger scale, a comprehensive and inexpensive framework to assess microbial freshwater communities. The project is centred around Oxford Nanopore Technologies' portable nanopore sequencing device, the MinION (Urban & Holzer et al., 2020).

© Lucinda Price

Developing a metagenomic sequencing workflow for freshwater monitoring

We benchmarked the design of essential experimental steps for multiplexing samples on the MinION (Olsen et al., 2016), ranging from water sampling to DNA extraction and library preparation. By including defined microbial compositions in our freshwater study, we evaluated computational methods including twelve taxonomic classification tools for the bacterial classification of nanopore sequencing reads from 16S rRNA-amplicon libraries. To showcase the resolution of sequencing-based freshwater monitoring in a spatiotemporal setting, we combined DNA analyses with physicochemical measurements at nine locations within a confined ~12 km reach of the River Cam at three different time points (April, June and August 2018).

Following our approach, we were able to resolve the core microbiome of the River Cam, as well as assess its temporal and spatial fluctuations (see Urban & Holzer et al., 2020, for all results). Besides common freshwater bacteria, nanopore sequencing allowed us to distinguish closely related pathogenic and non-pathogenic bacterial species by leveraging multiple sequence alignments between nanopore and reference reads. For example, the Leptospira genus contains several saprophytic species (obtaining energy from dead and/or decaying organic matter) and several intermediate species (of unknown pathogenicity), but also the disease-causing agents of Leptospirosis, a life-threatening waterborne infection that is known to occur in the UK in small numbers every year. Despite the presence of nanopore sequencing errors and therefore inflated read divergence, our multiple sequence alignment approach allowed us to pinpoint a distinctly higher similarity between our nanopore reads and saprophytic rather than pathogenic Leptospira species. The standard qPCR workflow of Public Health England to assess Leptospiral infections confirmed the presence of saprophytic species, suggesting that the current nanopore sequencing quality is sufficiently high to yield indicative results for bacterial monitoring at the species level (Urban et al., 2020).

In general, we found an enrichment of potentially pathogenic bacteria downstream rather than upstream of (or within) the boundaries of Cambridge. Interestingly, we found the peak of Leptospira abundance to fall into an area of increased sewage influx from a nearby wastewater treatment plant; an enrichment of Leptospira bacteria in wastewater effluents had already been described by others, including researchers in Berlin, Germany (Numberger et al., 2019).

The benefits of MinION for metagenomic sequencing in the field

Compared to previous sequencing machines, the MinION dramatically reduces financial and time investment, and is deployable during fieldwork applications due to its portability. In addition, the long-read sequencing technology of the MinION allowed us to sequence the entire bacterial 16S rRNA region, simultaneously tagging various hypervariable regions, and therefore allowing for deeper taxonomic resolutions than short-read approaches. While we estimated the error rate of our study to be 8%, and found systematic errors such as increased indel rates at homopolymer reference sites, clustering of the nanopore reads with known reference reads enabled us to resolve certain bacterial compositions down to the species level and to describe significant shifts of human pathogen candidates along an urban river trajectory.

Using an easily adaptable and scalable framework centred around the MinION sequencing device, we were able to assess the spatio-temporal microbiome of a freshwater system at an expense of less than £4,000 (Urban & Holzer et al., 2020). To inform the general public about the merits of genomic research, we engaged in various outreach activities, set up a comprehensive website including an introductory video, and organised a workshop to teach our entire framework – from water sampling to computational analyses – to interested scientists and citizens. We hope that this project will stimulate more environmental monitoring initiatives and health organisations to use this inexpensive, portable framework, to ultimately increase the proportion of citizens that benefit from genomic research.

Credit: Orla Moore

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