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.
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
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.
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
Bawn M
(2022)
Single-cell genomics reveals population structures from in vitro evolutionary studies of Salmonella.
in Microbial genomics
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 developed an approach for single bacterial genomic analysis using FACS isolation of individual bacteria followed by whole-genome amplification and sequencing. We apply this to in vitro experimental evolution of a hypermutator strain of Salmonella in response to antibiotic stress (ciprofloxacin). By analysing sequence polymorphisms in individual cells from the population we identified the presence and prevalence of sub-populations which have acquired polymorphisms in genes previously demonstrated to be associated with ciprofloxacin susceptibility. This population structure could not be resolved from bulk sequence data, and our results show the applicability of high-throughput single-cell sequencing to experimental studies of bacterial evolution. |
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 |
URL | https://www.biorxiv.org/content/10.1101/2020.08.25.266213v1 |
Description | We have a collaboration with Sphere, and Onix to develop their microfluidic platforms for microbial sequencing. There is a growing interest in these application from industry and our work in this area has led to collaborations with other industry partners in using these tools for string the human microbiome - one of these companies is now sponsoring a PhD student at EI in this area. We plan to develop these tools further to offer a service for microbial single cell analysis through our NBRI |
First Year Of Impact | 2022 |
Sector | Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
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 | Membership of BBSRC Transformative technology Panel |
Geographic Reach | National |
Policy Influence Type | Membership of a guideline committee |
Impact | The transformative technology strategy advisory panel have influence BBSRC policy on data intensive bioscience and big ideas pipeline |
Description | Darwin Tree of Life |
Amount | £9,360,421 (GBP) |
Funding ID | 218328/Z/19/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 11/2019 |
End | 05/2023 |
Title | Method for sequencing individual microbial cells and mapping SNP to reference genome data |
Description | We have developed a method for sequencing individual bacterial cells and happy the resulting data to reference genome data to phase SNPs and identify sub populations growing within planktonic cuture. |
Type Of Material | Technology assay or reagent |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | This has been used to identify the impact of low concentration antibiotics on mutation rate. |
URL | https://www.biorxiv.org/content/10.1101/2020.08.25.266213v1 |
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 | 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 |