Trying to identify DNA methylation mutations with current technologies usually requires a lot of cells, making it a lot like hunting for a needle in a haystack. Getting down to looking at single cells is becoming possible, but requires you to look at the whole genome, making the hunt for epimutations difficult. Recent work from Silvia Gravina and Jan Vijg from the Albert Einstein College of Medicine in the Bronx, NY have found a way to narrow the search.
These researchers wanted to reduce the cost in the world of single-cell bisulfite sequencing by finding a way to focus on a specific region. They developed a new approach which they call Single cell, Locus-specific Bisulfite sequencing (SLBS) to cut the scale and cost of single cell DNA methylation analysis.
After painstaking optimization and proof-of-concept experiments, a protocol emerged robust enough to reliably find epimutations at specific loci in single cells.
Here are the basics of the protocol, and some of the interesting findings from their proof-of-concept experiments:
- Single cells are lysed and bisulfite treated.
- DNA is immediately whole-genome amplified.
- PCR using primers specific to regions of interest are used for sanger sequencing.
- SLBS was able to show cell-to-cell variation in DNA demethylation in a 5-Aza-time course experiment.
- SLBS detected an epimutation event in in the Cyp71a promoter in a single neuron of four analysed, and event traditional sequencing would have missed.
- Using SLBS to estimate the epimuation rate for the first time, the authors found it to be 2.7%, 2 orders of magnitude higher than the DNA mutation rate.
SLBS could have applications in examining cell-to-cell diversity or finding cancer epimuations. There are a lot of ways to make looking for a needle in a haystack easier; SLBS sifts out the hay, making the tiny epimuation needles easier to find.
Get the full report in Nucleic Acids Research, April 2015