text: Massively parallel single molecule direct RNA-Seq has been explored as an alternative to traditional RNA-Seq, in which RNA-to-cDNA conversion, ligation, amplification, and other sample manipulation steps may introduce biases and artifacts. Technology platforms that perform single-molecule real-time RNA-Seq include Oxford Nanopore Technologies (ONT) Nanopore sequencing, PacBio IsoSeq, and Helicos (bankrupt). Sequencing RNA in its native form preserves modifications like methylation, allowing them to be investigated directly and simultaneously. Another benefit of single-molecule RNA-Seq is that transcripts can be covered in full length, allowing for higher confidence isoform detection and quantification compared to short-read sequencing. Traditionally, single-molecule RNA-Seq methods have higher error rates compared to short-read sequencing, but newer methods like ONT direct RNA-Seq limit errors by avoiding fragmentation and cDNA conversion. Recent uses of ONT direct RNA-Seq for differential expression in human cell populations have demonstrated that this technology can overcome many limitations of short and long cDNA sequencing.