Understanding how genes are regulated requires more than simply measuring which genes are turned on or off. Researchers also need to know which RNA molecules are produced, how they are spliced, whether they carry chemical modifications, and how long their poly(A) tails are. Traditional RNA sequencing methods often answer only part of that puzzle.

Researchers at Xi’an Jiaotong University provide a comprehensive overview of nanopore direct RNA sequencing (DRS), highlighting how the technology is changing transcriptomics and epitranscriptomics.

Unlike conventional RNA sequencing, nanopore direct RNA sequencing reads native RNA molecules directly. Because the RNA does not need to be converted into DNA or amplified before sequencing, the technology preserves many of the molecule’s natural features. This allows researchers to examine full-length RNA transcripts while simultaneously detecting RNA modifications, alternative splicing events, and poly(A) tail lengths from individual RNA molecules.

Timeline of major innovations in RNA sequencing and RNA modification technologies

(A) Evolution of RNA modification analysis, from early chromatographic approaches (mid-20th century), to LC-MS/MS and RT-PCR-based methods (1980s), followed by transcriptome-wide antibody-based mapping (e.g., MeRIP-seq/m⁶A-seq, m⁶A-LAIC-seq) from 2012 onwards, and more recent enzymatic and chemical labeling strategies (e.g., DART-seq, MAZTER-seq, m⁶A-SEAL-seq, m⁶A-SAC-seq). Nanopore DRS with the SQK-RNA004 chemistry achieves enhanced performance and, when combined with Dorado software v1.0.0 and above, can detect up to eight distinct RNA modifications. (B) Development of RNA sequencing technologies, including Sanger sequencing (1977), next-generation sequencing (2000s), long-read platforms (e.g., PacBio RS; 2010s), and nanopore sequencing (from 2014). The introduction of Oxford Nanopore DRS from 2016 is indicated. DART-seq, deamination adjacent to RNA targeting sequencing; DRS, Direct RNA sequencing. LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry; m⁶A-LAIC-seq, N6-methyladenosine level and isoform characterization sequencing; m⁶A-SAC-seq, N6-methyladenosine site-specific alkali cleavage sequencing; m⁶A-SEAL-seq, N6-methyladenosine sequencing via enzymatic-assisted alkali ligation; m⁶A-seq, N6-methyladenosine sequencing; MAZTER-seq, MazF endonuclease-assisted RNA m⁶A profiling sequencing; MeRIP-seq, methylated RNA immunoprecipitation sequencing; PacBio RS, Pacific Biosciences real-time sequencing; RT-PCR, reverse-transcription polymerase chain reaction.

The researchers explain that this combination of information provides a much more complete picture of how genes are regulated. Rather than studying each RNA feature separately, researchers can investigate multiple layers of regulation within the same transcript, offering new insights into cellular function and disease.

The researchers also compare nanopore direct RNA sequencing with other sequencing approaches. Short-read RNA sequencing remains highly accurate and cost effective for many applications, while long-read cDNA sequencing provides excellent transcript reconstruction. Nanopore DRS complements these methods by preserving native RNA chemistry, making it especially valuable for studying the epitranscriptome and complex transcript structures.

The researchers also discusses practical considerations for implementing nanopore direct RNA sequencing. It summarizes optimized laboratory workflows for a wide variety of RNA molecules, including messenger RNA, ribosomal RNA, transfer RNA, circular RNA, microRNA, and non-poly(A) transcripts. In addition, the authors outline quality control recommendations and best practices for obtaining reliable sequencing data.

On the computational side, the researchers highlight recent advances in software that improve RNA modification detection, transcript reconstruction, isoform quantification, and poly(A) tail analysis. The authors emphasize the importance of distinguishing direct measurements from computational predictions and recommend validating important findings with complementary experimental methods.

Looking ahead, nanopore direct RNA sequencing is expected to play an increasingly important role in precision medicine, agriculture, plant biology, and animal research. As sequencing accuracy and computational methods continue to improve, direct analysis of native RNA may help researchers better understand disease mechanisms, discover biomarkers, and characterize increasingly complex transcriptomes.