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.

Many bacterial genes are regulated by RNA elements in their 5´ untranslated regions (UTRs). However, the full complement of these elements is not known even in the model bacterium Escherichia coli. Using complementary RNA-sequencing approaches, we detected large numbers of 3´ ends in 5´ UTRs and open reading frames (ORFs), suggesting extensive regulation by premature transcription termination. We documented regulation for multiple transcripts, including spermidine induction involving Rho and translation of an upstream ORF for an mRNA encoding a spermidine efflux pump. In addition to discovering novel sites of regulation, we detected short, stable RNA fragments derived from 5´ UTRs and sequences internal to ORFs. Characterization of three of these transcripts, including an RNA internal to an essential cell division gene, revealed that they have independent functions as sRNA sponges. Thus, these data uncover an abundance of cis- and trans-acting RNA regulators in bacterial 5´ UTRs and internal to ORFs.