While trying to uncover the mysteries of the DNA methylation, a lot of BS is often required. And no, we’re not talking about workplace politics but rather sodium bisulfite (BS) conversion. Thankfully, the labs of Chun-Xiao Song and Benjamin Schuster-Böckler at Oxford University (UK) have developed a new “BS-free” approach to uncover the secrets of the methylome called Tet-assisted pyridine borane sequencing (TAPS).
The motivation behind developing TAPS was two-fold. First, was the fact the sodium bisulfite is a harsh chemical treatment that degrades DNA. Second, sodium bisulfite converts unmethylated cytosines to uracil, which PCR then converts to thymine. Co-senior author Chun-Xiao Song notes that, “this is very inefficient and computation intensive. It’s like looking for Waldo by eliminating everyone who is not Waldo. TAPS allows us to find Waldo directly.”
Here’s how TAPS works:
- A ten-eleven translocation (Tet1) enzyme oxidizes both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) to 5-carboxylcytosine (5caC)
- Pyridine borane reduces 5caC to dihydrouracil, a uracil derivative that PCR converts to thymine
Aside from the standard version that detects both 5mC and 5hmC, TAPS comes in two other varieties:
- TAPSβ: β-glucosyltransferase labels 5hmC with glucose protects 5hmC from the oxidation and reduction reactions and allows for specific detection of 5mC
- Chemical-assisted pyridine borane sequencing (CAPS): Potassium perruthenate acts as a chemical replacement for Tet1 and specifically oxidizes 5hmC, thus allowing for direct detection
With their new sequencing approach in hand, the talented team then compared TAPS to whole-genome bisulfite sequencing (WGBS) by assaying mouse embryonic stem cells, where they found that TAPS:
- Doesn’t degrade DNA and thus preserves long molecules while achieving conversion rates similar to sodium bisulfite
- Produces higher sequencing quality scores for cytosines and guanine base pairs
- Provides a more even coverage of various genomic features (such as CpG islands)
- Doesn’t require a PhiX spike-in to balance the nucleotide composition of libraries
These advantages ultimately mean that TAPS provides higher alignment rates, which reduces sequencing costs. Finally, the team demonstrated that TAPS works with DNA inputs as low as 1 ng, including circulating cell-free DNA.
Co-senior author Benjamin Schuster-Böckler adds, “Compared to bisulfite sequencing, processing TAPS data is not only twice as fast, it also retains a lot more information from the original sample. This makes it much easier to detect mutations and structural variations even while identifying DNA modifications.”
Song concludes, “We think TAPS could directly replace bisulfite sequencing as a new standard in DNA epigenetic sequencing. It makes DNA epigenetic sequencing more affordable and accessible to a wider range of academic research and clinical applications. You can imagine that if you’re dealing with very limited genetic samples, such as cell-free DNA circulating in blood, bisulfite sequencing becomes very challenging.”
Go see how TAPS can take the BS out your methylomes over at Nature Biotechnology, February 2019