Metagenomic analysis of planktonic riverine microbial consortia using nanopore sequencing reveals insight into river microbe taxonomy and function

Abstract

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.

Freshwater monitoring by nanopore sequencing

“…Most routine freshwater surveillance frameworks focus on semi-quantitative diagnostics of only a limited number of target taxa, such as pathogenic Salmonella, Legionella and faecal coliforms ( Ramírez-Castillo et al, 2015 ; Tan et al, 2015 ), whereas metagenomics approaches can give a complete and detailed overview of environmental microbial diversity. Besides nanopore shotgun-sequencing ( Reddington et al, 2020 ), our proof-of-principle analysis highlights that targeted full-length 16S rRNA gene MinION sequencing is a suitable complement to hydrochemical controls in pinpointing relatively contaminated freshwater sites, some of which in case of the River Cam had been previously highlighted for their pathogen diversity and abundance of antimicrobial resistance genes ( Rowe et al, 2017 ; Rowe et al, 2016 ). Nanopore amplicon sequencing has here allowed us to reliably distinguish closely related pathogenic and non-pathogenic bacterial species of the common Legionella , Salmonella , Pseudomonas , and Leptospira genera.…”

“…Although there are still common concerns about the technology's base-level accuracy, mobile MinION setups have already been transformative for real-time tracing and rapid data sharing during bacterial and viral pathogen outbreaks ( Boykin et al, 2019 ; Chan et al, 2020 ; Faria et al, 2018 ; Faria et al, 2017 ; Kafetzopoulou et al, 2019 ; Quick et al, 2015 ; Quick et al, 2016 ). In the context of freshwater analysis, a MinION whole-genome shotgun sequencing protocol has recently been leveraged for a comparative study of 11 rivers ( Reddington et al, 2020 ). This report highlights key challenges which emerge in serial monitoring scenarios of a relatively low-input DNA substrate (freshwater), for example large sampling volumes (2–4 l) and small shotgun fragments (mean < 4 kbp).…”

Freshwater monitoring by nanopore sequencing

“…Our results show that it would be theoretically feasible to obtain meaningful river microbiota from >100 barcoded samples on a single nanopore flow cell, thereby enabling water monitoring projects involving large collections at costs below £20 per sample (Supplementary Table 6). On the other hand, shotgun nanopore sequencing approaches such as the one employed by Reddington et al (2020) may bypass pitfalls often observed in amplicon sequencing, namely taxon-specific primer biases (Frank et al, 2008), 16S rDNA copy number fluctuations between species (Darby, Todd, & Herman, 2013) or the omission of functionally relevant sequence elements. In combination with sampling protocol adjustments, shotgun nanopore sequencing could moreover be used for the serial monitoring of eukaryotic microorganisms and viruses in freshwater ecosystems (Reddington et al, 2020).…”

“…Since the commercial launch of the MinION in 2015, a wide set of microbial nanopore sequencing applications in the context rRNA gene (Benitez-Paez, Portune, & Sanz, 2016; Cusco, Catozzi, Vines, Sanchez, & Francino, 2018; Kerkhof, Dillon, Haggblom, & McGuinness, 2017; Krehenwinkel et al, 2019) and shotgun (Leggett et al, 2019; Nicholls, Quick, Tang, & Loman, 2019; Reddington et al, 2020; Stewart et al, 2019) metagenomics have attracted the interest of a growing user community. Two independent case studies have recently provided decomposition analyses of faecal bacterial pathogens in MinION libraries derived from river and spring waters in Montana, USA (Hamner et al, 2019) and Kathmandu Valley, Nepal (Acharya et al, 2019).…”

Microbial diversity characterization of seawater in a pilot study using Oxford Nanopore Technologies long-read sequencing

“…In this pilot study we evaluate the utility of Oxford Nanopore Technologies (ONT) sequencing to characterize microbial diversity in seawater. Our strategy is based on a method to analyze riverine samples [33] and aims to classify microbial diversi cation directly from environmental samples with minimal computational and nancial cost over a relatively short time span. This will facilitate future scalable investigations such as monitoring oceanic biodiversity and landscape the time and space dynamics these microbes are subject to.…”