cDNA and RNA sequencing: revealing the transcriptome

RNA, or ribonucleic acid, plays a central role in gene expression and regulation¹. By studying RNA molecules within a sample, researchers can investigate which genes are active, how transcripts are processed, and how these processes change in different biological conditions¹.

Changes in gene expression and RNA processing are associated with many diseases. For example, approximately 15% of hereditary diseases and cancers are associated with aberrant gene expression², making accurate transcript characterisation an important area of research.

Oxford Nanopore offers two approaches for transcriptome analysis: cDNA sequencing, which converts RNA into complementary DNA (cDNA) before sequencing, and direct RNA sequencing, which analyses native RNA molecules and preserves information about features, such as poly(A) tail length and RNA modifications.

In this Nanopore Know-How blog, we explore how direct RNA and cDNA sequencing work, the different insights each approach provides, and how researchers are using them to investigate the transcriptome.

Why is cDNA sequencing important for transcriptomics research?

Sequencing cDNA is the predominant method for investigating the transcriptome. In this technique, reverse transcription of RNA transcripts produces ‘copy’ cDNA molecules, which are then sequenced.

Short-read cDNA sequencing, often known as RNA-seq³, can capture splice sites and transcription start and end points⁴. However, because reads only cover small portions of RNA molecules, reconstructing complete transcripts and distinguishing closely related isoforms can be challenging⁵. Isoforms are different RNA transcripts produced from the same gene⁴ through processes such as alternative splicing, and understanding them is important for investigating gene regulation and disease².

So, how can nanopore cDNA sequencing capture complete transcript isoforms?

How does Oxford Nanopore technology sequence full-length transcripts?

The Oxford Nanopore cDNA sequencing method converts total RNA into double-stranded cDNA through reverse transcription and PCR, without fragmentation, before nanopore sequencing. Our cDNA-PCR Sequencing Kit has been designed to capture transcripts in full using unrestricted nanopore reads, and with the latest protocol update, we have improved the reverse transcription step to reduce internal priming. This is a known limitation of other cDNA technologies and enables accurate estimation of poly(A) tail length and captures a better representative view of the transcriptome than before.

More information about our cDNA-PCR Sequencing Kit (including the multiplexing version, the cDNA-PCR Barcoding Kit) and when to use it can be found in the masterclass ‘Which library prep workflow is right for my experiment?’. Plus, see our workflow overview for sequencing full-length transcripts for isoform-level expression analysis in bulk transcriptomic experiments.

Nanopore sequencing generates reads of any length, enabling isoform analysis without assembling short reads. For example, Heberle and Brandon et al. identified spliced RNA transcripts with nanopore cDNA sequencing, revealing novel full-length RNA isoforms associated with Alzheimer's disease and other neurological disorders⁴.

However, what if you could directly analyse RNA rather than a copy of RNA?

Can you directly sequence native RNA?

No longer are you limited to analysing just DNA or cDNA; Oxford Nanopore sequencing is the only technology that enables you to sequence RNA directly. By developing an RNA-specific motor protein and nanopore, you can now directly sequence native RNA, allowing you to analyse complete RNA transcripts.

For direct RNA sequencing, reverse transcription of total RNA generates a cDNA strand. This secondary strand improves stability and removes any kinks or secondary structures in the RNA, reducing pore blocking and increasing sequencing data yield — but only the RNA strand is sequenced. With unrestricted nanopore reads, complete RNA transcripts are captured, including splice junctions and poly(A) tails, so you can see the full diversity of isoforms in your samples.

More information about the Direct RNA Sequencing Kit, including our multiplex version, the Direct RNA Barcoding Kit, can be found in the masterclass ‘Which library prep workflow is right for my experiment?’. To see an overview of a complete RNA sequencing experiment, you can download our workflow overview for detecting isoforms and modifications with PCR-free, direct RNA sequencing.

What additional data do you get with direct RNA sequencing?

Direct RNA sequencing opens access to the epitranscriptome. As direct RNA sequencing does not require amplification or PCR, base modifications, such as methylation, are preserved. This means that you can capture both full-length isoforms and RNA methylation from the same sequencing data.

Currently, nanopore sequencing detects eight different RNA modifications simultaneously: N⁶-methyladenosine (m⁶A), inosine, 5-methylcytosine (m⁵C), pseudouridine (PseU), 2’-O-methyladenosine (2’-Ome-A), 2’-O-methylcytidine (2’-Ome-C), 2’-O-methylguanosine (2’-Ome-G), and 2’-O-methyluridine (2’-Ome-U), delivering a comprehensive view of the transcriptome and epitranscriptomic diversity.

Using this additional information, Wang et al. investigated epitranscriptomic modifications to research host immune responses to influenza virus exposure⁷. With direct RNA sequencing, they revealed ‘changes in viral and host RNA … at higher resolution than standard methods, enhancing our understanding of these processes’⁶.

Why is methylation important? Check out the methylation Nanopore Know-How blog to find out why it matters.

How are the Nanopore Community using RNA and cDNA sequencing?

As highlighted throughout, the Nanopore Community have been using RNA and cDNA sequencing to accelerate transcriptomics research across a range of human disease applications. An example is from Kim et al., who used direct RNA sequencing to simultaneously analyse the transcriptome and epitranscriptome in human leukaemia cell lines to investigate RNA features including m⁶A modifications, mRNA stability, and poly(A) tail length⁷.

Researchers have also used nanopore sequencing to overcome accessibility limitations in low-resource settings. Lin et al. used nanopore cDNA sequencing to streamline detection of gene fusions associated with B-cell acute lymphoblastic leukaemia (B-ALL). Current clinical methods require multiple complex tests, such as flow cytometry and karyotyping. With a novel gene fusion detection algorithm and their nanopore transcriptomic workflow, the team demonstrated that they could directly detect B-ALL-associated gene fusions as a single assay. Plus, as Oxford Nanopore sequencing uses a benchtop sequencer, the study demonstrates how nanopore sequencing is ‘highly adaptable across various resource settings’⁹. Watch Thomas Alexander, a co-author of this publication, present another study using transcriptome sequencing in low-resource settings.

Transcriptomics is about more than measuring gene expression. From resolving transcript isoforms with cDNA sequencing to investigating RNA modifications with direct RNA sequencing, Oxford Nanopore technology enables researchers to explore multiple layers of RNA biology. The best workflow depends on the questions you want to answer.

How will you take transcriptomics research beyond gene expression-level analysis?

Find out more about bulk transcriptomics sequencing with Oxford Nanopore with the getting started guide

Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.