7 breakthroughs in RNA research from the Nanopore Community

On RNA Day 2019 we're taking the time to celebrate some ground-breaking work from the Nanopore Community, across the field of transcriptomics. These examples utilise both methods we offer for long-read transcriptome analysis — cDNA and direct RNA sequencing:

  1. Annotation: Obtaining a four-fold increase in transcriptome diversity

Obtaining full length transcripts with nanopore long-read sequencing enhances de novo transcriptome assembly and novel isoform discovery. Anthony Bayega and team used full-length nanopore cDNA sequencing to obtain a four-fold increase in transcriptome diversity of the developing olive fly compared to the predicted NCBI transcriptome. You can hear from Anthony in this webinar or read a summary of his results here.

  1. Non-coding RNA: 62% of targeted haplotype blocks were transcribed into novel RNAs not described in GENCODE annotations

The majority of SNPs associated with neuropsychiatric traits reside in non-coding regions, potentially in non-coding RNA. Hardwick et al. used targeted RNA capture and long-read cDNA sequencing on the PromethION to investigate novel RNA expression in disease-relevant regions. Using this approach, the team were able to identify splicing between distant exons, and overall exhibited more uniform exon coverage than short-read sequencing. Read the full publication here.

  1. Transposable elements: enhanced repeat resolution with nanopore long-read cDNA sequencing

The repetitive nature of transposable elements makes them challenging to map with short sequencing reads. In this publication, Jiang et al. performed 5’Cap capturing of native RNA for nanopore direct sequencing of full-length transcripts, enabling the accurate identification of RNA spliceforms in the locust transcriptome.

  1. Gene fusions: MinION and Flongle cDNA sequencing enable fusion detection within hours

William Jeck and his team have developed a rapid, cost-effective method of detecting gene fusions, responsible for cancers such as acute leukaemia, using real-time cDNA sequencing on the MinION and Flongle. With the first fusion reads detected within "seconds" of sequencing, they demonstrated the potential of nanopore sequencing to dramatically reduce the crucial time-to-treatment for such diseases in future.

  1. Single-cell: resolving antigen receptor sequences at nucleotide resolution with nanopore long reads

Traditional high-throughput single-cell sequencing is limited to short-read sequencing of the end of cDNA molecules. To enable full resolution of transcripts at the single-cell level, Mandeep Singh et al. developed a method combining long nanopore reads and single-cell sequencing of lymphocyte transcripts. Studying samples from a breast cancer patient, they resolved antigen receptors "at nucleotide resolution" and investigated lymphocyte clonal evolution. Read the full publication here.

  1. Modifications: detecting m6A in native RNA with nanopore direct RNA sequencing

Oxford Nanopore provides the only technology which enables direct sequencing of native RNA, without conversion to cDNA. Making use of this, Eva Maria Novoa and her team have developed an accurate method of detecting m6A modifications, shown to be pivotal to processes including cell differentiation and stress response, in native RNA.

  1. RNA viral genomes: sequencing the first complete RNA virus in its native form with 100% nucleotide coverage

Keller et al. harnessed Direct RNA nanopore sequencing to sequence the coding-complete Influenza A virus genome: the first example of sequencing an RNA virus genome in its native form. Reaching 100% nucleotide coverage and 99% consensus identity, they noted that their method could be adapted to sequence viral mRNA, (+) sense cRNA and other viral pathogens. Read the full publication here.

Happy RNA Day!

Find out more about RNA and gene expression analysis using direct RNA and cDNA sequencing.