Sometimes, you have to jump through a lot of hoops to get what you want. Hanlee Ji’s lab (Stanford) must be tired of playing those games because they’ve developed a direct single-molecule methylation nanopore sequencing approach for cell-free DNA (cfDNA) that doesn’t involve bisulfite conversion or even PCR. And yet it works on nanogram or smaller amounts of DNA—ideal for analyzing very low levels of cell-free DNA (cfDNA) from cancer patient samples.
To detect methylation on cfDNA shed by tumors into the bloodstream, you usually have to play the bisulfite conversion game to change unmodified cytosines to uracils, then do PCR and short-read sequencing. That means a lot of steps and possibly introducing artifacts and damaging DNA. Many approaches also need a lot of DNA for this to work, which is just not feasible with the low levels of cfDNA in samples from real patients.
So, tossing out the usual gameplan of PCR and bisulfite conversion, this team developed a new approach—one that incorporates an improved nanopore-based single-molecule sequencing technique and directly provides data for the DNA in its native state:
- Add barcodes and nanopore sequencing adapters using the team’s optimized end-repair, a-tailing, and ligation conditions
- When multiplexing, perform a second end-repair and a-tailing step, adding new Ji-lab-developed sequencing adapters
- Sequence with Oxford Nanopore PromethION system and analyze the data with “megalodon” software
Compared to the traditional nanopore sequencing protocol, the new strategy increased the aligned read yields by an order of magnitude, and could handle amounts as low as nanogram and picogram levels. When analyzing cfDNA from colorectal cancer patients and healthy controls, the new method revealed that the cancer patient samples:
- Were more varied in their genome-wide methylation levels
- Had more mononucleosomes
- Exhibited more methylation differences in genes and promoters
Then, with a little computation, the team determined a classification score and determined whether individual cfDNA reads originated from tumor or immune cells.
In a final checkmate move, the cfDNA of three patients with different gastrointestinal cancers were analyzed. Methylation data from the samples taken from the patients over time correlated with their treatment histories. When a treatment worked, tumor-specific methylation changes decreased; when the cancer began to spread again, the team saw these changes increase.
No hide-and-seek here—if you want more details on this new technique, read more at Genome Medicine, May 2023.