Date: 3rd April 2023
Calling methylation in the human genome with PCR-free nanopore sequencing
Methylation plays a fundamental role in regulating gene expression; aberrant methylation patterns are strongly associated with numerous diseases, such as cancer and developmental disorders. Using traditional short-read sequencing technology, epigenetic modifications are erased during PCR, so must instead be inferred via chemical treatment of DNA such as bisulfite conversion. However, this process can give variable results due to incomplete conversion, and cannot distinguish between methylation variants 5mC and 5-hydroxymethylcytosine (5hmC).
With PCR-free nanopore sequencing of native DNA, methylation can be directly detected at single-nucleotide resolution. Base modifications can be accurately called alongside the canonical nucleotide sequence, with no additional sample preparation, including in genomic regions that may be inaccessible to traditional sequencing methods; furthermore, both 5mC and 5hmC modifications can be called.
Here we present a simple workflow for genome-wide methylation calling from a human blood sample, using the PromethION platform.