Long-read nanopore cDNA sequencing and direct DNA methylation detection resolves copy number debate in Cannabis

DNA methylation plays an essential role in governing the architecture of gene expression across eukaryotes. In plants, DNA methylation not only protects the genome from transposable element (TE) jumping by repressing their expression, it also mediates environmental-specific expression responses.

Here Todd presents how he used direct DNA methylation detection coupled to full-length cDNA sequencing across several Cannabis chemotypes (high and low THC/CBD) to understand the final step in the cannabinoid pathway. The final step of the cannabinoid pathway is the Tetrahydrocannabinol acid synthase (THCAS) and/or Cannabidiol acid synthase (CBDAS), which are intron-less genes that are nested in 50-80 kb TE units tandemly duplicated at several loci in the genome.

Due to the complexity of the THCAS/CBDAS loci and the fact that they are highly similar, full-length cDNA sequencing was required to resolve which synthases were expressed in different chemotype genomes.

Furthermore, short-read based bisulfite sequencing fails to distinguish the DNA methylation patterns at these loci. However, direct DNA methylation enables the characterization of DNA patterns across these complex loci, providing evidence that DNA methylation in part explains the observed expression patterns.