Development of single-cell sequencing technology for microbial populations

Lead Research Organisation: Earlham Institute
Department Name: Directorate Office

Abstract

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.

Technical Summary

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

Planned Impact

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.

Publications

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Richards TA (2019) Single cell ecology. in Philosophical transactions of the Royal Society of London. Series B, Biological sciences

 
Description we are able to successfully sort single bacterial cells, amplify and sequence them.
We have undertaken in vitro evolution studies with salmonella in weak antibiotic selection and demonstrated that we see antimicrobial resistance mutations arising. Uniquely we are able to resolve haplotypes of individual lineages
Exploitation Route Our results could be used by us or others to study:
understanding the emergence of antimicrobial resistance.
understanding biofilm sub-functionalisation
understanding evolutionary processes
Sectors Environment,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description BBSRC ENWW panel membership
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
Impact The panel advises BBSRC exec and councillor how best to deliver BBSRC strategy. Impact is difficult to quantify
 
Description Chair of BBSRC Institute Partnership
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description Oral Evidence to House of Commons Science and Technology Committee enquiry on Balance and effectiveness of research and innovation spending
Geographic Reach National 
Policy Influence Type Gave evidence to a government review
URL https://parliamentlive.tv/Event/Index/4bdf3ff7-d1a6-4605-8b73-aca355bf9f89
 
Description submission to UKRI infrastructure review
Geographic Reach National 
Policy Influence Type Participation in a national consultation
URL https://www.ukri.org/files/infrastructure/progress-report-summary-document-final-march-2019-pdf/
 
Description EI-QIB collaboration in microbial genomics 
Organisation Quadram Institute Bioscience
Country United Kingdom 
Sector Academic/University 
PI Contribution There is a joint PDRA post in this area and now a joint PI group. EI is undertaking bioinformatics development and single cell and genomic analysis. We also provide HPC capability.
Collaborator Contribution The team at QIB are undertaking the evolutionary experiments, providing samples and data. We work closely with the Webber and Kingsley groups in this area.
Impact The collaboration involved microbiology and bioinformatics.
Start Year 2015
 
Description Darwin Tree of Life Project (UK) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release detailing the institutes involvement (partner) in the project which will create a new foundation for biology to drive solutions for preserving biodiversity and sustaining human societies. The institute is likely to be involved in the sampling and sequencing of protists (single-celled eukaryotes - organisms with a nucleus) and the analysis of vertebrate genomes.
Year(s) Of Engagement Activity 2018
 
Description Decoding Life on Earth - The Earth BioGenome Project (EBP) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Press release detailing the institutes involvement (partner) in the project which is a global effort to sequence the genetic code of all 1.5 million known animal, plant, protozoan and fungal species on Earth. The project will create a new foundation for biology to drive solutions for preserving biodiversity and sustaining human societies. The aim is to sequence, catalogue and categorise the genomes of all of Earth's eukaryotic biodiversity over a period of ten years.
Year(s) Of Engagement Activity 2018
 
Description Feature article on genomics in the Easton Daily Press 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The Article was to cover the research activity at the Earlham Institute and at the Norwich Research park and how it would impact the general public
Year(s) Of Engagement Activity 2019
 
Description Interviewed for a BBC news feature on the Darwin Tree of LIFE project - shown worldwide on BBC world news 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact The feature covered the Darwin Tree of Life project and the involvement of EI. Particularly focussing on Protists
Year(s) Of Engagement Activity 2019
 
Description Just one more Ash Dieback spore could push European ash trees to the brink 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact News article on disease
Year(s) Of Engagement Activity 2018
 
Description One day in New York: bioinformatics and family life 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Article on work life balance and career journey/progression
Year(s) Of Engagement Activity 2018
 
Description Phone Interview with Tom Chivers for article about how "algorithms are changing genetic research" for the publication Mosaic 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Phone Interview with Tom Chivers for article about how "algorithms are changing genetic research" for the publication Mosaic. The article was subsequently published online in the Independent and Spectrum.
Year(s) Of Engagement Activity 2018
URL https://www.independent.co.uk/news/long_reads/big-data-biology-cancer-science-genome-sequencing-chro...
 
Description Techology Day at Norwich Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 7500 members of the general public attended Nature Day at Norwich Science Festival. A number of activities were available to engage the general public with the work of the institute. A number of talks/presentations were scheduled also.
Year(s) Of Engagement Activity 2018
 
Description What doesn't kill you makes you stronger? Cracking Salmonella and antibiotic resistance 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Article on tackling Salmonella by exploring a highly mutable, evolved strain to help answer why antibiotics are becoming less effective and improve our bodies' antimicrobial armour.
Year(s) Of Engagement Activity 2018