Complex microbiomes are everywhere. Our skin, our guts, soil, pond water. All are home to tiny life forms, interacting with each other and going about their business.
There are estimated to be at least a trillion species of microorganisms populating our planet, and 99.9 per cent of them are still undiscovered.
Sorting and sequencing the microscopic members of these communities sounds like a daunting task - but it’s one Yash Bancil, a third-year PhD student in the Macaulay lab at the Earlham Institute, has been devoting his time to.
Yash using the Atrandi microfluidic technology for high-throughput single-cell analysis
His project involves using the Institute’s innovative single-cell technologies to develop methods for unravelling the genomic and functional complexities of very different microbiomes.
He is using the Institute’s cutting-edge technology, such as flow cytometry and droplet-based microfluidics.
Yash is a CASE student with the Norwich Research Park Doctoral Training Partnership (NRPDTP), meaning he collaborates with an industry partner on his project.
He is working with Microbiotica, a biopharmaceutical company specialising in the development of precision live biotherapeutic products.
His work also benefits from the Institute’s single-cell and spatial analysis specialist facilities, part of our Transformative Genomics National Bioscience Research Infrastructure (NBRI).
“My PhD is all about using single-cell technologies to explore complex microbiomes,” explains Yash. “I’m developing methods which can be used generally across different microbiomes - so I’m looking at several totally different environments.
“I’ve analysed soil and pond water, and my current collaboration with Microbiotica means I have access to microbes derived from the human gut.
“In the past, we’ve used bulk methods to sequence microbiomes, but this approach can result in missing valuable insights - rare species or cellular heterogeneity may go unnoticed.
“Single-cell approaches provide a potential way to explore every cell in a microbiome without losing any of the data.”
He says researchers in the microbiome field are becoming increasingly interested in identifying rarer species in complex microbial communities since they may act as keystone species.
“For example, the bacteria Porphymonas gingivalis is known to be present in patients with periodontitis. It’s found at very low abundance, but the part it plays in the microbiome makes patients more susceptible to disease progression.”
I’m looking at ways to tease apart complex microbial communities. I have managed to separate out different types of bacteria from each other and enrich for rare species, allowing me to look at them in a lot more detail.
PhD researcher Yash Bancil
Single-cell sequencing of microbiomes can give researchers the chance to study some rare species in greater detail for the first time.
“I’m looking at ways to tease apart complex microbial communities,” says Yash. “I have managed to separate out different types of bacteria from each other and enrich for rare species, allowing me to look at them in a lot more detail.”
He says possible applications could include identification of specific pathogens or biomarkers quickly in complex samples.
“Using a high-throughput screening method like this you could potentially do that very quickly,” he says.
Yash is using two technologies to help develop his broad microbiome analysis technique. The first is flow cytometry with spectral sorting on the BD FACSDiscover S8.
“This equipment uses the basic principles of flow cytometry, but uses up to 78 fluorescent detectors to show fluorescence across a larger spectrum of wavelengths. When cells are fluorescently labelled, they can be detected,” he says.
“The spectral detection runs from UV light to red light, generating a lot of data. This is also the first instrument to perform high-speed image-enabled cell sorting and it generates images of each cell or particle passing through at an impressive 15,000 events per second.
“By using both fluorescence and cell morphology, I can sort cells of interest into a 96- or 384- well plate. Sorted microbial cells are lysed, and femtogram amounts of DNA can be amplified to produce sequencing libraries.”
The second arm of his research is droplet-based microfluidics, using the Onyx platform from Atrandi Biosciences.
Single cells are isolated and encapsulated in semi-permeable capsules. These are used to compartmentalise single-cell reactions - a technique which improves DNA amplification and helps with identifying cellular heterogeneity, making it much more efficient for reconstructing whole genomes.
Below: the process of a single microbial cell being isolated using the Onyx platform
We’re picking up things that were not seen before but were always there. As well as enriching for rare species, we’re able to sort for functionality, associate mobile genetic elements with more precision, and analyse bacterial interactions - for instance with bacteriophages.
Both methods help Yash dissect and study microbial communities, although he is keen to stress these techniques need to be used in tandem with metagenomics.
“Bulk sequencing and single-cell sequencing are complementary techniques and you have to use both to get a full picture of a particular microbiome” he says.
“They both provide useful information and should be used alongside each other.”
He says single-cell sequencing opens up insights into previously unseen communities and interactions.
“We’re picking up things that were not seen before but were always there,” says Yash. “As well as enriching for rare species, we’re able to sort for functionality, associate mobile genetic elements with more precision, and analyse bacterial interactions - for instance with bacteriophages.
“I’ve also been able to isolate fungal components of microbiomes. These are often overlooked, as bacteria are generally easier to study, but a microbiome is a collection of microorganisms - including fungi, algae, archaea and protists.
It’s really important for the field to look at these as well, as they all contribute to the functional activity of a microbiome.”
After taking a degree in biochemistry from the University of Bath in 2018, Yash worked as a research assistant for Microbiotica. He found the human microbiome fascinating, and it wasn’t long before he decided to head back into academia. He took an MSc at King’s College London before joining the Earlham Institute for his PhD programme.
