Workflow: direct RNA sequencing
Detecting isoforms and RNA modifications with PCR-free, direct RNA nanopore sequencing
Accurately capturing the range of RNA diversity — including gene expression, isoforms, and RNA methylation — can help elucidate the molecular mechanisms of disease and functional roles of RNA modifications. For example, RNA modifications, such as n6-methyladenosine (m6A), impact RNA metabolism and play a significant role in initiation and progression of cancers, as well as neurological disorders, including Alzheimer’s disease and Huntington’s disease.
Nanopore sequencing is the only available technology that directly reads native RNA transcripts, enabling quantitation of gene and isoform expression without PCR bias. It also provides resolution of full-length isoforms and accurately estimates poly-A tail length. Furthermore, direct RNA nanopore sequencing provides direct detection of m6A methylation for the DRACH motif at single-base resolution without additional sample preparation.
Here we present a simple workflow for RNA modification analysis from a human blood research sample, using direct RNA sequencing on PromethION.