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Capturing Droplets in Beads, Epic-PCR Enables Massively Parallel Single-Cell Sequencing

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NEW YORK (GenomeWeb) – A team led by researchers at the Massachusetts Institute of Technology has developed a method to link phylogeny and function in each of the millions of individual cells in a microbial community with a single high-throughput experiment.  

The droplet-based technique is called Emulsion, Paired Isolation, and Concatenation PCR, or epic-PCR.

In a proof-of-principle study published last week in the International Society of Microbial Ecology Journal, the researchers demonstrated that epic-PCR could be used for high-throughput investigations of conserved genetic traits in hundreds of thousands of cells at once, at a cost similar to preparing one genomic library.

Keys to the method are the creation of polyacrylamide bubbles, or beads, around single cells suspended in oil-emulsion droplets and the use of concatenated PCR to query two loci in each genome.

"We add this little surface of a hydrogel, sort of like a plastic, around each one of the cells; the plastic keeps the cells together, so once you use your strongest methods to break open the cells the DNA won't float anywhere because the gel matrix keeps it in place," Manu Tamminen, a corresponding author on the study, told GenomeWeb.

The hydrogel matrix is dense enough to hold bacterial genomes inside after bead recovery, but loose enough to allow enzymes and primers to diffuse through, so "each one of your cells gets a completely separate, private reaction compartment," he said.

The method then creates a concatemer of the functional target gene and the phylogenetic marker gene and uses a PCR step with three primers such that, "if there is an empty bubble or if it has a genome that does not have your target genes, then the PCR will not take off and there will be no amplification," Tamminen explained.

This step is then followed by nested PCR to re-amplify the concatenated product and also add the adapters for Illumina MiSeq sequencing. Tamminen said the MiSeq gives a nice read length for this sort of application but that he is also considering using Thermo Fisher IonTorrent or Oxford Nanopore MinIon sequencing in future work.

In the ISME study, Tamminen and his colleagues used lysosome and proteinase K to bust open bead-trapped bacteria that had been dredged from the depths of Upper Mystic Lake.

This Boston-area freshwater lake has the notoriously-polluted Aberjona river as a tributary, and its proximity to MIT means it has been the subject of numerous studies. Tamminen and his colleagues thus already knew that the bottom of the lake lacked oxygen and was depleted of other electron acceptors so that bacteria had to rely on sulfate.

"We also had a good idea of what sort of genes for sulfate reduction we should be able to amplify by PCR down there; we didn't know exactly what to expect but we had a good way of testing the sanity of our results based on previous studies," Tamminen said.

The team queried the dissimilatory sulfate reductase gene, or dsrB, from all 16S ribosomal RNA-containing cells. In this way they could link function with phylogeny on the single-cell level to answer the question of 'who is doing what' in the lake-bottom bacterial community. They confirmed the presence of bacteria they'd anticipated, but also discovered previously unknown diversity.

Using polyacrylamide hydrogel to encapsulate cells is not new, nor is metagenomic sequencing, but connecting the two via emulsion PCR seems to be original.

"Let's say not that much has been done around it so far — which is surprising because once you get around certain technical hurdles it's not even technically that demanding," Tamminen said.

Alternatively, metagenomics on whole communities is "practically impossible" if one wishes to look at a rare gene in a population at the single-cell level, he said. "You will never find a fast enough computer to crunch all of those sequences and give you an answer, [and] there are some methodological things that get in the way."

Tamminen believes the method could be adapted to other types of cells, and the team is investigating using barcoding similar to methods described in two recent Cell papers covered by GenomeWeb.

"Barcoding of total DNA in one bubble would lead to something that would sort of bridge the disciplines of metagenomics and single-cell genomics — it would provide the throughput of metagenomics with the precision of single-cell genomics," Tamminen said.

The polyacrylamide bubbles can also be modified in future applications, by "for instance add[ing] PCR primers ... or some other molecules that give new chemical properties," Tamminen said, adding, "I think there is a lot of potential there, some of which we have already explored, but a lot of potential that we haven't even realized yet."

Tamminen is now at the Swiss Federal Institute of Technology in Zurich, but he continues this work in collaboration with the lab of Eric Alm at MIT, including the graduate student who was first author on the ISME study, Sarah Spencer. They are pursuing using epic-PCR for studies of antibiotic resistance and biogeochemical cycles. "We are still working very hard together to take this in some new directions," Tamminen said.

And while the epic-PCR method itself is intriguing, being able to profile bacterial communities for traits such as sulfate reduction at a single-cell resolution is also scientifically important.

"If you think about it in a wider context, cycles of sulphur, carbon, phosphorus, and nitrogen are the great elemental cycles of our planet that basically keep everything from halting to a thermodynamic equilibrium, and therefore understanding those on a global scale is of huge importance and relates to basically everything that humans do," said Tamminen. 

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