cDNA and RNA sequencing: revealing the transcriptome

RNA, or ribonucleic acid, is present in nearly all organisms. It serves many functions, including protein synthesis and gene regulation¹. There are many different types of RNA, such as messenger RNA (mRNA), which is transcribed from the genes in the DNA, and used by ribosomes to build proteins¹.

To study gene expression, researchers analyse mRNA via copy or complementary DNA (cDNA) sequencing to reveal more about normal cell functioning and disease aetiology¹. Approximately 15% of hereditary diseases and cancers are associated with aberrant gene expression², meaning that accurately identifying and annotating isoforms will expose new disease biology.

Unlock the transcriptome with Oxford Nanopore technology. In this Nanopore Know-How blog, we explore how nanopore reads of unrestricted length span full-length transcripts to reveal more than before through RNA and cDNA sequencing.

Why is cDNA sequencing important for transcriptomics research?

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

Legacy short-read cDNA sequencing, often known as RNA-seq³, can capture splice sites and transcription start and end points⁴. However, as RNA-seq generates short reads, the method cannot accurately characterise complete transcripts nor identify isoforms. Isoforms are RNA transcripts from the same gene that vary only slightly. They are generated by alternative splicing, a gene regulation process that increases protein diversity; however, when alternative splicing is unregulated, diseases can result². Therefore, it is critical to confidently characterise isoforms to understand the impact of aberrant splicing events — a challenge with short reads⁴.

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. 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: How to select the right library prep workflow for your experiment.

Nanopore sequencing generates reads of any length, which can span the full length of transcripts from RNA. This means that you can easily analyse transcript isoforms without the need to assemble short reads, as demonstrated by Oikonomopoulos et al., who noted that the ‘full-length sequencing of the cDNA molecules permits the unambiguous assignment of isoforms’⁵.

cDNA sequencing with Oxford Nanopore technology has uncovered many novel isoforms that legacy technologies previously missed. 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 disorders⁶.

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 directly sequence RNA. 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.

More information on this process can be found in the masterclass: How to select the right library prep workflow for your experiment, along with a detailed walkthrough of the end-to-end direct RNA sequencing workflow.

What additional data do you get with direct RNA sequencing?

Direct RNA sequencing opens up your 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 achieve both full-length isoform resolution and RNA methylation detection from the same sequencing data.

Utilising 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’⁷.

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. Another 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 length⁸.

Other researchers have also compared direct RNA nanopore sequencing with legacy cDNA short-read sequencing techniques. He, Ganesamoorthy, and Chang et al. sequenced RNA from whole human blood research samples with sepsis infections, comparing the Oxford Nanopore Direct RNA Sequencing Kit with Illumina cDNA sequencing⁹. While both platforms provided robust gene expression data, they found that Oxford Nanopore sequencing ‘offers unique advantages not provided by Illumina cDNA-sequencing’ because nanopore technology uncovered post-transcriptional modifications and ‘revealed numerous novel isoforms through Nanopore direct RNA-seq’⁹.

Researchers have also used nanopore sequencing to overcome limitations in low-resource settings. Opoku et al. achieved whole-transcriptome analysis of solid tumour research samples with cDNA sequencing on a MinION. The team stated that with further development, the method has the ‘potential to eliminate the need for stepwise testing and increase access to diagnostic tools in resource-constrained settings, helping to bridge the existing cancer diagnostic gap’¹⁰*. Watch Thomas Alexander, a co-author of this publication, present a similar study using transcriptome sequencing in low-resource settings.

Isoform-level expression is not only available for bulk transcriptomics, but also at single-cell resolution — find out more about single-cell sequencing in a future blog!

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.