Highly parallel direct RNA sequencing on an array of nanopores

Abstract

Ribonucleic acid sequencing can allow us to monitor the RNAs present in a sample. This enables us to detect the presence and nucleotide sequence of viruses, or to build a picture of how active transcriptional processes are changing – information that is useful for understanding the status and function of a sample. Oxford Nanopore Technologies’ sequencing technology is capable of electronically analysing a sample’s DNA directly, and in real-time. In this manuscript we demonstrate the ability of an array of nanopores to sequence RNA directly, and we apply it to a range of biological situations. Nanopore technology is the only available sequencing technology that can sequence RNA directly, rather than depending on reverse transcription and PCR. There are several potential advantages of this approach over other RNA-seq strategies, including the absence of amplification and reverse transcription biases, the ability to detect nucleotide analogues and the ability to generate full-length, strand-specific RNA sequences. Direct RNA sequencing is a completely new way of analysing the sequence of RNA samples and it will improve the ease and speed of RNA analysis, while yielding richer biological information.

The RNA modification landscape in human disease

“…3A). Initially limited to DNA sequencing, ONT published their first results of direct RNA sequencing at the end of 2016 (Garalde et al 2016). Several months later, a direct RNA-sequencing (DRS) kit finally became available to the general public in April 2017 (Fig.…”

“…There have nonetheless been recent reports describing computational models capable of detecting modified DNA bases, by training models from biological control data and by observing conspicuous alterations of ionic current at specific positions (Stoiber et al 2016;McIntyre et al 2017;Rand et al 2017;Simpson et al 2017). With respect to DRS, these strategies have recently been applied to characterize the epitranscriptome, namely the identification of m 6 A (Garalde et al 2016) and conserved 16S ribosomal RNA base modifications and a 7-methylguanosine modification associated with antibiotic resistance . It is likely that, following the release of the direct RNA-sequencing kit, additional algorithms to detect and predict RNA modifications from DRS data will become available.…”

Avian transcriptomics: opportunities and challenges

“…The first avian transcriptomes have recently been sequenced using this new technology (Kuo et al 2017;Workman et al 2017). Another new application, which enables sequencing of full-length transcripts, is the MinION from Oxford Nanopore Technologies (Ayub et al 2013;Garalde et al 2016). However, to our knowledge this application has not yet been used for RNA sequencing in bird species.…”

“…Furthermore, a number of errors, such as self-priming, template switching, and reverse transcriptase inaccuracies can occur during cDNA synthesis (Ozsolak and Milos 2011;Wang et al 2009). Also, most current RNA-seq protocols contain a templateamplification step, and as certain transcripts might be more prone to amplification than others, this step can lead to the uneven distribution of transcripts which is not representative of the sample (Garalde et al 2016;Kozarewa et al 2009). New technologies are continuously being developed to avoid or minimise these issues, including long-read technologies (Gonzalez-Garay 2016), direct RNA sequencing technologies (Ayub et al 2013;Garalde et al 2016;Ozsolak et al 2009), and PCR-free protocols (Mamanova et al 2010).…”

“…Also, most current RNA-seq protocols contain a templateamplification step, and as certain transcripts might be more prone to amplification than others, this step can lead to the uneven distribution of transcripts which is not representative of the sample (Garalde et al 2016;Kozarewa et al 2009). New technologies are continuously being developed to avoid or minimise these issues, including long-read technologies (Gonzalez-Garay 2016), direct RNA sequencing technologies (Ayub et al 2013;Garalde et al 2016;Ozsolak et al 2009), and PCR-free protocols (Mamanova et al 2010). Ensuring a high -quality library is of highest priority in all NGS projects, and it is therefore likely that we will see further developments aimed at minimising the problems involved in library preparation for RNA-seq within the near future.…”