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Yes. Nanopore direct RNA sequencing reads native RNA molecules without first sequencing a DNA copy. The pore measures changes in electrical current as RNA passes through it, and software infers the nucleotide sequence from that signal. This is different from translating a genetic code: translation interprets a coding RNA sequence to determine a protein, while sequencing identifies the RNA’s bases.
What “reading the genetic code directly from RNA” means
Here, “reading” means determining an RNA molecule’s nucleotide sequence, not translating its codons into amino acids. RNA uses A, U, G and C; DNA uses T instead of U. After sequencing, a coding RNA sequence can be interpreted to identify the protein it encodes, but that is a separate step.
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In direct RNA sequencing, the molecule sensed by the instrument is native RNA rather than a reverse-transcribed DNA copy. “Direct” does not mean sample preparation is absent: the cited Oxford Nanopore workflow uses enzymes, adapters and cleanup steps.
How a nanopore reads an RNA molecule
Oxford Nanopore describes its flow cell as a membrane containing nanopores, each connected to an electrode and sensor channel. As a molecule passes through a pore, it changes the ionic current. The changing signal—often called a squiggle—is processed by basecalling algorithms to infer the sequence. Oxford Nanopore’s sequencing overview explains this sensing principle.
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In the SQK-RNA004 workflow, RNA moves through the pore in the 3′ to 5′ direction, while the basecaller presents the resulting read in the 5′ to 3′ orientation. The complementary DNA strand made during preparation helps stabilize the RNA and improve sequencing output; it is not the strand sequenced. Oxford Nanopore’s protocol puts it this way: “The complementary cDNA strand is not sequenced, but improves the RNA sequencing output.”
What direct RNA sequencing can reveal
Because the instrument senses native RNA, features of the original molecule—including some base modifications—can affect the measured signal. This distinguishes the approach from ordinary cDNA sequencing, which reads a converted DNA copy rather than directly measuring the original RNA. Oxford Nanopore describes these capabilities in its RNA library preparation overview.
That does not mean every modification is automatically identified or that every sample has a validated modification result. Detection depends on signal analysis and the methods used; the vendor’s general capability statements do not establish sensitivity, specificity or accuracy for every modification or sample.
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| Consideration | Direct RNA sequencing | cDNA-based RNA sequencing |
|---|---|---|
| Molecule sequenced | Native RNA passes through the pore. | A DNA copy made from RNA is sequenced. |
| Original RNA modifications | May affect the native-RNA signal and can be relevant to modification analysis. | Does not directly measure the original RNA molecule in the same way. |
| Amplification and bias | Can be useful when reduced PCR-amplification bias matters. | Preparation may involve amplification, which can introduce bias. |
| Difficult-to-reverse-transcribe transcripts | May be useful when reverse transcription is a concern; the SQK-RNA004 workflow nevertheless makes a stabilizing cDNA strand. | Requires reverse transcription to make the DNA template. |
| Output and workflow | Requires a compatible RNA kit, flow cell, device and prepared RNA sample. | Oxford Nanopore says its cDNA kits may offer higher output per run when direct-RNA advantages are not needed. |
The output comparison is Oxford Nanopore’s product characterization, not an independent head-to-head result. Neither method is universally better: the choice depends on whether measuring native-RNA features and reducing amplification-related bias are worth the workflow trade-offs. See the vendor’s Direct RNA Sequencing Kit information for its product description.
Independent reader supportYour contribution helps us test, update, and keep practical guides available for everyone.What the SQK-RNA004 laboratory workflow involves
Oxford Nanopore’s SQK-RNA004 protocol describes a research-laboratory workflow using poly(A)-tailed RNA or total RNA. The protocol estimates approximately 85 minutes for reverse transcription, 45 minutes for adapter ligation and cleanup, and 10 minutes for flow-cell priming and loading. These are protocol estimates, not guaranteed hands-on times.
- Prepare and check RNA. Start with poly(A)-tailed RNA or total RNA and assess sample length, quantity and purity using appropriate RNA quality-control supplies.
- Make a stabilizing strand. Synthesize complementary cDNA to support the RNA during sequencing; the cDNA is not sequenced.
- Prepare the library. Attach sequencing adapters and clean up the library according to the kit protocol.
- Prime and load the flow cell. The protocol lists MinION/GridION RNA flow cells (FLO-MIN004RA) and PromethION RNA flow cells (FLO-PRO004RA) as compatible.
- Acquire and basecall data. Use MinKNOW for data acquisition and basecalling as specified in the protocol.
The kit is not a complete sequencing system. The workflow also requires a compatible MinION, GridION or PromethION device, the matching RNA flow cell, an RNA sample, quality-control supplies and laboratory equipment such as pipettes and a thermal cycler. Oxford Nanopore marks the protocol For Research Use Only; it does not establish the method as a clinical test or a sequence read as a diagnosis.
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