On the design and prospects of direct RNA sequencing

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Abstract

Throughout the past nearly a decade, the application of high-throughput sequencing to RNA molecules in the form of RNA sequencing (RNA-seq) and its many variations has revolutionized transcriptomic studies by enabling researchers to take a simultaneously deep and truly global look into the transcriptome. However, there is still considerable scope for improvement on RNA-seq data in its current form, primarily because of the short-read nature of the dominant sequencing technologies, which prevents the completely reliable reconstruction and quantification of full-length transcripts, and the sequencing library building protocols used, which introduce various distortions in the final data sets. The ideal approach toward resolving these remaining issues would involve the direct amplification-free sequencing of full-length RNA molecules. This has recently become practical with the advent of nanopore sequencing, which raises the possibility of yet another revolution in transcriptomics. I discuss the design considerations to be taken into account, the technical challenges that need to be addressed and the biological questions these advances can be expected to resolve.

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