Alternative splicing increases the complexity of the human transcriptome as multiple transcripts can be generated from a single gene. However, short-read RNA-seq are limited in their ability to resolve complex splicing patterns and rely on conversion of RNA into cDNA. Long-read methodologies that generate reads spanning entire transcripts have great potential for identifying splice isoforms. Oxford nanopore direct RNA (dRNA) sequencing is an emerging method with great potential for un-biased expression profiling but it’s ability to detect differential expression of genes and isoforms has not been established.
To address this, dRNA sequencing was performed on undifferentiated and differentiated (neuron-like) SH-SY5Y cells with addition of RNA spike-ins as a positive control. Overall, ~4 million aligned native RNA reads with a median aligned length of 988 nucleotides were generated, identifying 20,158 genes and 42,206 transcripts, as well as 1,384 novel isoforms. Analysis of spike-in controls confirmed highly accurate expression quantification.
Differential expression was successfully detected between undifferentiated and differentiated 5Y cells, identifying 304 (p-value<0.05) differentially expressed genes between cell types. Gene ontology analysis identified upregulation of the axon guidance pathway during differentiation, confirming the biological relevance of the identified expression changes. These results show that dRNA sequencing can reliably detect spliced isoforms, quantitate gene expression and identify differentially expressed genes. This validates the ability of dRNA sequencing to identify biologically relevant changes in gene expression and its applicability to better characterise isoforms involved in development and disease.