Development of single-cell sequencing technology for microbial populations

When biological samples are analysed at the DNA level (to study genotypes) or the RNA level) to look at gene expression. We currently have to analyze many thousands or millions of cells at a time. This has been a very informative approach but it means that we miss a lot of complexity in biological systems, as in most cases the DNA in a microbial culture may be different in each cell and the gene expression in each cell is also hetrogeneous. This is particularly important is communities that form biofilms on surfaces as there may be different strains or species of bacteria working together performing different roles in the community. Biofilms are especially important for bacteria evading antimicrobial treatment.
In this project we aim to develop the methodologies for single cell sequencing of microbes and test these against a number of different use cases. We will use Fluorescence Activated Cell Sorting (FACS) to separate cells and then develop protocols to analyze these cells in parallel by using molecular barcoding methods.

Next-generation sequencing has enabled routine sequencing of bacterial isolates for national epidemiological surveillance programs is becoming more common. This has led to significant advances in relating bacterial pathogenicity to disease etiology and their combined impact to human health and food security. Standard sequencing techniques, however, rely on the bulk sequencing of cultured samples and as such do not fully resolve the genetic heterogeneity of bacterial communities. High-throughput single-cell sequencing approaches have the potential to rectify this and elucidate the mechanisms operating within these communities as they evolve under varying modes of selection. However, the majority of single-cell technology to date has focussed on eukaryotic systems, and solutions dedicated to microbial systems remain an unmet need.

The long term aim is to establish methods to observe the genotypes and transcriptional phenotypes of mixed microbial populations including heterogeneity of genotype from clonally derived populations under selection, and the transcriptome heterogeneity arising from naturally occuring epigenetic mechanisms. The proposed experiments are based on preliminary data that indicate that defined mixed species populations can be sorted by FACS for conventional single-cell DNA sequencing. By developing FACS-based methods for microbial isolation along with combinatorial indexing and sequencing, we will enable robust, high-throughput approaches for bacterial analysis. We will use microbial mock community samples for technology development but aim subsequently to address short, but related Biological Objectives to demonstrate the real-world applicability of the methods develop. We will examine genomic heterogeneity in hypermutator strains of Salmonella enterica under selectionunder selection from subinhibitory concentrations of antibiotics give rise to heterogeneity of transcription within populations of Salmonella

Industrial Impact: The methods developed here will be of interest to a wie rage of industrial end users including those working in pharmaceutical and agrochemical development, in medical and agricultural diagnostics as well as in personal care and household products that seek control microbial growth. EI and QIB work with a large number of industry partners including Unilever, ABagri, DANONE and Syngenta who work in this space and with whom we will actively investigate potential collaborations in this area.
Also these techniques will be developed within the BBSRC funded national Capability for Genomics and Single Cell analysis and it is our intention to make these methods available to the wider scientific community via fee for service.


Societal Impact: EI and QIB actively engage with the general public to inform them about our science and engage with discussion about its potential impact. This work will focus on two areas of science that have a major public interest at the moment; Microbiomes and antibiotic resistance. To ensure that we disseminate the importance of this study we will work with our KEC team to produce publicity and social media interest. We already undertake a many engagement events such as open days and we will also write blogs and engage with the community though social media platforms such as twitter.