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 enable transcripts to be sequenced end-to-end, enabling their simple, accurate quantification and 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.
Introduction
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 three nanopore sequencing techniques (PCR-cDNA, direct 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 (see poster).
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 amplification-free direct cDNA and direct RNA sequencing kits, 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 (see poster), providing more precise insights into gene expression. Three streamlined RNA sequencing kits are available from Oxford Nanopore, each of which offers high sequencing yields from low input amounts (Table 1).
Table 1: Oxford Nanopore provides three distinct RNA sequencing methodologies, including PCR-free and reverse transcription-free approaches.
Case study
Transcription dynamics of the developing olive fruit fly embryo
Using full-length nanopore cDNA sequencing, Bayega et al. examined the isoform complexity and transcriptional dynamics of the early embryonic stages of the olive fruit fly (Bactrocera oleae) — one of the most important pests of cultivated olive trees. Sex determination in these organisms is determined within the first 6 hours of embryonic development. Through examining differential gene expression at specific timepoints during this key developmental stage, the team aim to develop novel sex-based pest control methods.
‘We generated a de novo transcriptome assembly of the olive fly and identified 3553 novel genes and a total of 79,810 transcripts; a four-fold increase in transcriptome diversity compared to the NCBI predicted transcriptome.’
Bayega et al.
Sequencing workflow
How do I perform gene expression analysis using nanopore sequencing?
Oxford Nanopore provides three streamlined RNA sequencing kits that can be used for gene expression analysis, all 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 Bioinformatics section of the Nanopore Community for data analysis tutorials.
Discover more about the advantages of full-length nanopore RNA sequencing for gene expression analysis and read customer case studies.
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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.
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