Combining Nanopore direct RNA sequencing with genetics and mass spectrometry for analysis of T-loop base modifications across 42 yeast tRNA isoacceptors.

Transfer RNAs (tRNAs) contain dozens of chemical modifications. These modifications are critical for maintaining tRNA tertiary structure and optimizing protein synthesis. Here we advance the use of Nanopore direct RNA-sequencing (DRS) to investigate the synergy between modifications that are known to stabilize tRNA structure.

Advances in nanopore direct RNA sequencing.

Nanopore direct RNA sequencing (DRS) reads continuous native RNA strands. Early adopters have used this technology to document nucleotide modifications and 3′ polyadenosine tails on RNA strands without added chemistry steps. Individual strands ranging in length from 70 to 26,000 nucleotides have been sequenced.

Nanopore ReCappable sequencing maps SARS-CoV-2 5' capping sites and provides new insights into the structure of sgRNAs.

The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5' cap, thus preventing reliable identification and quantification of full-length, coding transcript models.

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing.

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional direct RNA nanopore sequencing, the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites.