Gene expression

Analysis of gene expression is important in many applications, from clinical research to developmental biology. However, the use of traditional short-read RNA-Seq technologies requires fragmentation of RNA samples and subsequent computational assembly, which can cause multi-mapping and limit quantification accuracy; this can be further limited by PCR bias. In contrast, long nanopore reads allow transcripts to be sequenced end-to-end, enabling accurate quantification and complete characterisation of isoforms in a single dataset. PCR-free library preparation options eliminate PCR bias, whilst direct RNA sequencing enables the simultaneous detection of base modifications.

  • Accurately characterise and quantify full-length transcripts at the isoform level using long sequencing reads
  • Generate high sequencing yields from low input amounts
  • Explore epigenetic modifications through direct RNA sequencing and eliminate bias with PCR-free approaches

Isoform-level gene expression analysis

Traditional RNA-Seq gene expression studies typically utilise sequencing reads of 50–100 nucleotides. While such short reads allow gene-level expression analysis, they preclude the differentiation of transcript isoforms, which, importantly, can exhibit different expression levels and functional properties (Figure 1). With nanopore sequencing, read length is equal to RNA (or cDNA) fragment length, allowing the unambiguous analysis of full-length transcripts — enabling accurate characterisation and quantification of gene expression at the isoform level (Figure 1). Using nanopore sequencing, full-length transcripts in excess of 20 kb in length have been generated. Find out more about splice variation.

Figure 1: Alternative splicing can give rise to numerous mRNA isoforms per gene, which in turn can alter protein composition and function. The short reads generated by traditional RNA-Seq techniques lose positional information, making the correct assembly of alternative mRNA isoforms challenging. Long nanopore RNA sequencing reads can span full-length transcripts, simplifying their identification and quantification.

Figure 2: Sequencing workflows that incorporate amplification are vulnerable to sequence-specific biases. Yeast transcriptome libraries were prepared using two nanopore sequencing techniques (PCR-cDNA and direct RNA) and a typical short-read cDNA technique. In all cases, GC bias in the nanopore data sets was lower than in the short-read data set.

Unbiased gene expression analysis

Research shows that PCR-amplified libraries tend to exhibit reduced complexity when compared to the total mRNA pool. Not all transcripts amplify with the same efficiency, causing drop-out of some RNA molecules and excessive amplification of others. These issues can be exacerbated by the use of traditional short-read sequencing technologies, which are known to exhibit GC bias, where sequences with low or high levels of GC content are underrepresented. In addition to offering a low bias PCR-based cDNA sequencing kit, Oxford Nanopore provides the amplification-free Direct RNA Sequencing Kit and a direct cDNA sequencing protocol, which have been shown to provide more accurate representation and quantification of the total mRNA pool, with less GC bias than traditional short-read RNA-Seq approaches (Figure 2). The unique facility of nanopore technology to directly sequence native RNA also enables the retention and analysis of base modifications, providing even more comprehensive gene expression data.

High yields of full-length transcripts

The long, full-length transcript reads delivered by nanopore sequencing technology have been shown to reduce the total number of reads required for differential gene expression when compared with traditional short-read sequencing technologies. For example, Bayega et al., reported that 40x fewer nanopore cDNA reads were required to detect the same number of genes as short-read technology. Furthermore, the longer read lengths provided by nanopore sequencing reduce multimapping, whereby a single read maps to more than one location in the genome or transcriptome, providing more precise insights into gene expression. Two streamlined RNA sequencing kits are available from Oxford Nanopore, both of which offer high sequencing yields from low input amounts (Table 1).

RNA and cDNA sequencing kit comparison

Table 1: Oxford Nanopore provides two distinct RNA sequencing kits. In addition, a protocol for direct, PCR-free sequencing of cDNA is available using the Ligation Sequencing Kit.

Case study

Discovery of novel neuropsychiatric disorder risk-gene transcripts in the human brain

Genome-wide association studies have identified hundreds of risk loci for neuropsychiatric disorders; however, due to the limitations of traditional short-read sequencing techniques, the contribution of alternative RNA splicing and transcript isoform expression to disease risk remains poorly understood. Ricardo De Paoli-Iseppi from The University of Melbourne, Australia, demonstrated how long nanopore sequencing reads enabled comprehensive transcript characterisation for 31 neuropsychiatric risk genes. Using the GridION device, the team identified 929 transcript isoforms, 775 (>83%) of which were novel. Many of these novel transcripts were shown to be highly expressed, providing exciting new avenues of research into neuropsychiatric disorders.

Our results show that current gene transcript annotations are incomplete and supports the use of long-read sequencing to identify novel RNA isoforms.

Ricardo De Paoli-Iseppi, The University of Melbourne, Australia

Sequencing workflow

How do I perform gene expression analysis using nanopore sequencing?

Oxford Nanopore provides two streamlined RNA sequencing kits that can be used for gene expression analysis, both of which deliver full-length transcripts with low GC bias. The choice of kit depends on your specific study requirements, including sample amounts, requirement for sample multiplexing, base modification detection, and desired number of reads.

Nanopore sequencing is uniquely scalable to suit your throughput and yield requirements, from the portable Flongle* and MinION devices to the flexible, higher throughput GridION and PromethION platforms.

A number of robust tools are available for analysing full-length nanopore RNA sequencing reads, both from the Nanopore Community and Oxford Nanopore. Visit the EPI2ME web page for information about Oxford Nanopore's intuitive, best practice data analysis pipelines.

*The Direct RNA Sequencing Kit and associated flow cells are currently not available for Flongle devices.

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Differential gene expression analysis for low sample numbers

The MinION device in conjunction with the cDNA-PCR Sequencing Kit and MinION Flow Cells typically delivers 7–12 million reads, making it suitable for differential gene expression analysis of low sample numbers. Sample multiplexing can be achieved using the PCR Barcoding Kit.


cDNA-PCR Sequencing Kit

Differential gene expression analysis pipeline


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