Imma Perfetto
Cosmos science journalist
A high-tech upgrade is allowing scientists to peer into the genetics of millions of individual cells, while achieving significant reductions in cost compared to traditional approaches.
The South Australian Genomics Centre (SAGC) has become the first certified service provider in the Asia-Pacific for Parse Bioscience’s Evercode technology.
The Evercode platform tags a unique “barcode” onto the RNA of individual cells within a complex sample. The level at which genes are expressed in each cell can then be determined using SAGC’s MGI DNBSEQ-T7 sequencer, the fastest and most-efficient sequencing platform in Australia.
This technique, known as “single-cell RNA sequencing” (scRNA-seq), reveals crucial insights into the different cell types, states, functions, and disease signatures within a sample.
Dr Munir Iqbal, a genomics staff scientist in single cell and spatial transcriptomics at SAGC, tells Cosmos that unlike traditional droplet-based methods which rely on expensive, specially designed microfluidics chips, the single-cell Evercode kits do not require investment in high-end instruments.
“It comes as a packed kit … barcodes are loaded in your normal standard 96-well plates. All the reagents are in a tube. The only thing you need is a lab benchtop and a multi-channel pipette,” he explains.
SAGC Partnerships Manager, Joel Bathe, says the new approach has significantly reduced the cost to carry out scRNA-seq.
“In 2 years we’ve gone from … profiling a single cell for 50 cents, now we’re at less than 10 cents – 7 cents is what I calculated in our last project. Research dollars are finite. So, if you can [sequence] 5 times the amount of sample with the same dollar, the scale of these projects is just going up, which is great.”
The SAGC was founded in 2020 to support genomics research across Australia. When researchers in the biological sciences need to sequence the genetic information within cells – including DNA, RNA, and epigenetics – they call on SAGC.
“With DNA, you want to accurately read the DNA. Quantification is less important because each cell has a complete set of the genome,” explains Bathe.
DNA, packaged away in chromosomes within the cell’s nucleus, encodes the genetic instructions to make proteins. RNA acts like a messenger to carry this information outside of the nucleus, where it can read by ribosomes to make proteins.
“With RNA, quantification is really important because you want to understand when genes are being turned up and turned down,” says Bathe.
Traditionally, the level of gene expression would be measured in bulk, capturing all the messenger RNA (mRNA) in a sample. But bulk RNA sequencing (RNA seq) can only reveal the average gene expression across all the cells.
“RNA-seq is good, it’s still very standard technology. But it masks the effect of low expressing genes,” says Iqbal. It also cannot reveal granular detail such as how cell types within a sample express genes differently to one another either.
“Now we can do it at a single cell level,” says Bathe. “You can see that, ‘oh, this type of cell is over expressing this particular gene.’
He adds that rare cell types, which might only make up 0.1% of a sample, are also very much of interest. “[But] if you’re only looking at 10,000 cells, which was an amazing breakthrough [for scRNA-seq] just 3 years ago, those numbers just aren’t going to be enough.”
The Evercode technology uses an approach called “split-pool combinatorial barcoding” to enable SAGC to now run anywhere from a few thousand cells to 5 million.
It works like this.
First, as many as 96 samples are loaded onto a 96-well plate. To each well a unique molecular “barcode” is then added, which attaches to the surface of all the cells and tags them as belonging to that sample. The samples are then pooled together, and the cells are redistributed across another 96 well plate. Each well contains a second unique barcode which is added on top of the first – like links in a chain.
Just these 2 rounds results in 9,216 (96 x 96) possible combinations of barcodes, making it possible to individually identify up to that many cells. If the process is repeated once more this number jumps to 884,736. After 4 rounds the potential unique combinations reaches almost 85 million.
The cells are then broken apart to release the mRNA, which attaches to the ends of the barcodes on the cell’s surface. This barcode, which can be read during sequencing, marks the mRNA as having originated from that cell.
But there’s a problem. Unlike sturdy double-stranded DNA, RNA is single stranded. “It’s not stable. It degrades so quickly. It’s temperature sensitive,” says Iqbal.
“What happens is, we are still studying RNA, but not physically putting RNA on the sequencer.”
Instead, an enzyme (reverse transcriptase) converts the single stranded mRNA into complementary DNA (cDNA), which encodes the same information but in double-stranded DNA form.
This cDNA can then be amplified using polymerase chain reaction (PCR) so that there is enough material to sequence. The cDNA is fragmented into more manageable lengths, and “adaptors” are added to the ends of the strands so that the fragments can be read by the sequencing technology.
SAGC has already supported projects from institutions across Australia and New Zealand using this platform.
“Each and every research group has its own biological question,” says Iqbal. “One is working on an aspect of finding cancer markers. The other is working to find the drug targets for a particular disease. A third group is working to see the immunology response to a certain vaccine.
“The core of the technology does not change.”
SAGC Centre Manager, Dr Sen Wang, adds: “We’re giving researchers the tools to interrogate biology at an unprecedented level – one cell at a time.”