The art of being single: advanced single-cell sequencing technology

“Our group focuses on the development and implementation of new ways to explore the molecular and functional identity of individual cells,” explains Macaulay. “To explore a cell’s molecular identity, we have established platforms and protocols for single-cell genomics, transcriptomics and multi-omic analysis. 

“Generally, we’re interested in extracting as much information as possible from each single cell we analyse. To do this, we’ve developed and established a set of multi-omic tools which let us read out genomic, epigenomic and transcriptomic information from the same cell.”

The group also continues to integrate new approaches – such as long-read sequencing – into single-cell workflows. “This has been a really exciting area of research,” says Macaulay, “allowing us to explore alternative splicing in individual cells. 

“Biologically, this is really important but it’s something that is overlooked in most current single-cell experiments, and we were fortunate to be awarded a BBSRC New Investigator Award to support this work.”

Macaulay insists that any data the Group has generated using this approach has been really eye-opening - showing multiple isoforms of the same gene expressed in individual cells, sometimes with profound functional implications for the role of that gene in the cell’s biology. 

“Having our labs co-localised with Karim Gharbi’s Genomics Pipelines team has really helped us to develop and automate these approaches, and lets us explore how we can share these new methods with the community quickly as demand emerges,” he adds. 

“The beauty of single-cell approaches is they can be applied across the spectrum of living systems, and so have potential benefits for a very wide research community. 

“A major focus of our efforts in the area has been the translation of these approaches from ‘traditional’ biomedical research to broader applications.” 
 

Single-celled organisms are, naturally, perfect candidates for single-cell sequencing. Accessing genomic information from individual bacteria, for example, is allowing the team to explore evolutionary processes as the population adapts to antibiotics. 

One area where this is incredibly important is in understanding how antimicrobial resistance (AMR) emerges. 

To tackle this, Dr. Johana Hernandez and Dr. Matt Bawn worked with the single-cell group, as part of a collaboration between EI and the neighbouring Quadram Institute, to establish experimental and computational tools to sequence the entire genomes of individual Salmonella. They used this data to extract mutational signatures associated with adaptation to antibiotics and were able to build a ‘family tree’ of the bacterial population as it evolved. 

Bawn, a Postdoctoral Scientist at EI and the Quadram Institute, says: “This is giving us an unprecedented snapshot of how AMR can emerge. 

“Although we profiled just a few hundred bacteria, we saw several sub-populations - including some which had already acquired one or two mutations known to enable resistance. 

“This has really changed how we’ll approach these questions in future, and we’re now looking at ways to profile thousands - or hundreds of thousands - of bacteria in a single experiment.”

Beyond this, methods developed in the lab have been adapted to study biodiversity at single-cell resolution. 

Protists are single-celled organisms which are neither plant, animal or fungus, yet they make up a huge proportion of Earth’s biomass and biodiversity. As part of the Darwin Tree of Life project, EI has used parallel Genome and Transcriptome sequencing - or G&T-seq - from the same single cell to profile the DNA and RNA of individual protists.

“The ability to unlock this kind of information from single protists - using methods originally developed with cancer biology in mind - enables us to explore their diversity in a whole new way,” explains Professor Neil Hall, Director of EI and one of the scientists leading the Darwin Tree of Life project. 

“We can start to think about generating annotated reference genomes from individuals within a population. This will give us a unique insight into the complexity of protist communities and protist functional genomics.”

The Earlham Institute’s capability in single cell genomics has been built over the past six years, with support for infrastructure totalling over £2m coming through three rounds of capital funding from BBSRC.

“We really started from scratch back in 2016,” recalls Macaulay. “With Ashleigh Lister, the first member of the group, we started by setting up the basics - tissue culture, microscopy and manual single-cell RNA-seq library preparation.”

With support from Sarah Cossey, EI’s Director of Operations, they secured BBSRC capital funding for a BD FACSMelody cell sorter, which was a real turning point in the development of the platform. 

“We were able to undertake cell sorting experiments in-house,” explains Macaulay, “meaning we could now routinely isolate anything from single bacteria to blood stem cells.

“This was a hugely empowering investment and created the opportunity to undertake method development and automation efforts, as well as pilot projects much more easily. It also enabled much greater engagement in collaborative work across Norwich Research Park.  

“In parallel, we also implemented single-cell protocols on the 10X Genomics Chromium. At the time, it was in heavy use as a platform for genome assembly using synthetic long reads, and the single-cell protocol was something of a side line. This has of course changed dramatically since then - the 10X is one of our most-used platforms nowadays! 

This early period was all about laying foundations. “Bringing in these key pieces of equipment allowed us to be more self-sufficient and build some momentum around single-cell at EI,” says Macaulay. 

“With the support of the EI Training team, we also held our first Single-Cell Symposium, which we have since run annually. It has become a cornerstone in our connection with the wider research community.” Coupled with the advanced scientific training on offer at EI, there has been an ever-growing list of people considering working with EI to explore single cell approaches.

The evolution of single cell genomics at EI is not just about cutting-edge technology. The passion and ambition of the people in the team has been the key to its success. 

“We’ve managed to grow a really enthusiastic and talented team,” says Macaulay. “Through the NRP DTP programme, as well as a collaborative grant funded by CRUK, we currently have three PhD students, with core and responsive mode BBSRC funding supporting two Research Assistants and two Postdoctoral Scientists.

“We’ve been fortunate enough to get collaborative funding with researchers at UEA (David Monk), the Roslin Institute (Tim Connelley and Musa Hassan) and the IRCCS Ospedale San Raffaele in Milan (Giovanni Tonon), which has allowed us to establish really productive research networks with a diverse set of communities, united by their interest in advanced single-cell analysis. 

“We’ve also had opportunities to help members of the team grow their careers - Ashleigh Lister joined our group as Research Assistant and has been fundamental to the platform’s development. Now a Senior Research Assistant, her wealth of expertise benefits our students, as well as visitors from other groups and institutes. 

“Edyta Wojtowicz joined us on her Henry Wellcome fellowship and has taken immense strides towards independence in her research career, commencing her junior group leader post at EI in early 2023.” 

The Single Cell Symposium has also been critical in building and mobilising the community. Coupled with the advanced scientific training on offer at EI, there are an ever-growing list of people considering single cell approaches.

The success of single sequencing and analysis at EI is, like so many scientific success stories, the result of long-term support, the right mix of expertise and infrastructure, and a team science approach that is helping Macaulay and his colleagues to realise the enormous potential of this technology.

“We know the platform can have even more impact on local and national science,” says Macaulay, “and we will soon recruit a platform manager. This role will support internal and external users as they engage with us and enable them to make the most of our capability.

“This will hopefully allow us to support even more successful grant and fellowship applications, and really help build a community of single-cell researchers based around EI.” 

The group is also keen to undertake further engagement with industry. 

“In 2020 we performed single-cell sequencing of mouse liver cells with Cytiva (formerly GE Healthcare) to perform a comparative analysis of cell dissociation methods,” explains Macaulay. 

“The data was used in support of a subsequent product release. We know there is a need from industry for support with single-cell genomics and we aim to be available to address these needs”