Methods and Protocols to Make Your Life Easier (4): Single Cell Sequencing

(January 17th, 2014) According to Nature Methods the sequencing of DNA and RNA of single cells is the hottest technique at the moment. Steven Buckingham tells you all you need to know about the Method of the Year 2013.

We’ve all been going on about how advances in sequencing have been speeding up the pace of research. But from time to time something happens that makes you realise it is not just about speed – sometimes, the steady march of technological advances opens up fundamentally new ways of asking biological questions.

Single-cell sequencing is just what it says on the tin. Take a single cell, amplify up the DNA and sequence it. Until now, the countless difficulties and pitfalls that come with amplifying DNA up from a single cell has meant single-cell sequencing has been left to a handful of experts. But more and more kits have been coming onto the market, bringing the new technique to the masses. There isn't one sudden technological breakthrough behind it all, just a constant evolution and refinement of what we already have, picking off the pitfalls of single-cell sequencing one by one.

Take the problem of allele dropout, for instance. Amplifying a single cell's genome runs the risk of missing out an allele altogether. To get around this, a trick called “multiple displacement amplification” can be used. The target DNA is incubated in a mixture of random primers, a polymerase enzyme and fluorescent markers to monitor the progress of the reaction. The primers bind to complementary sequences on the target DNA and the polymerases do their job of running off the cDNA. When the polymerases hit the adjacent stretch of primer, complementation continues, peeling off as a branch of DNA. More primers bind to that branch, and so the process repeats itself. The result is a highly branched strand of DNA, which is then deep-sequenced. 

All the same, some ingenious methods have played a role in elevating single-cell sequencing to the status of a factory process. A striking example is MIDAS (microwell displacement amplification system). This process uses an array of thousands of microwells, with each well having a volume of a few nanolitres. A fluid sample containing cells is pipetted into the plates, and the concentration of cells is adjusted such that by the laws of statistics, there will nearly always be at most one cell in any well. Progress is monitored by the intensity of fluorescent markers added in the amplification mix – and of course only the wells that happen to have a cell light up.

So what does single cell sequencing bring to the table? First, it means we can stop losing all that valuable information that we have been throwing away using traditional methods. Doing it the old way, DNA for sequencing was extracted from whole tissues or whole organisms, averaging out all the individual differences at the cellular level. Gone are the days when we used to believe that all the cells in an organism had exactly the same genome. Attention can now turn to understanding these cellular differences.

And remember, these differences can also be of practical use in their own right. For instance, an application that is gaining a lot of attention is high definition cell lineage tracing. At each cell division, there is a finite chance of random mutations occurring. These are inherited cumulatively, leaving a convenient genealogical record. Reading the genealogical record in the genomes of individual cells opens up new dimensions in understanding cell fate.

Then of course you have no choice but to use single-cell sequencing with cells and organisms that just can't be cultured for the usual DNA extraction methods. Circulating cancer cells, for instance, may only occur in small numbers in a blood sample. Single-cell sequencing of individual circulating cancer cells can answer some very fundamental questions about how metastasis arises – do cancer cells arise from any subpopulation of tumour cells, or from a limited subset? Do metastases sometimes arise after chemotherapy because a few cells survive treatment, or because metastatic cells arise from a dormant pool of stem cells that are untouched by chemo?

Then think of epigenomics. The transcriptomes of individual cells can be correlated, for instance, with their position in the tissue of origin. As with all new techniques, there is plenty to feed the imagination.

No wonder more than one commentator is predicting that we are at the beginning of a new focus on the single cell.

Steven Buckingham

Picture: Chris Schlag

Last Changes: 02.28.2014

Information 4

Information 5

Information 6