Project description:Sequencing was performed to assess the ability of Nanopore direct cDNA and native RNA sequencing to characterise human transcriptomes. Total RNA was extracted from either HAP1 or HEK293 cells, and the polyA+ fraction isolated using oligodT dynabeads. Libraries were prepared using Oxford Nanopore Technologies (ONT) kits according to manufacturers instructions. Samples were then sequenced on ONT R9.4 flow cells to generate fast5 raw reads in the ONT MinKNOW software. Fast5 reads were then base-called using the ONT Albacore software to generate Fastq reads.

2019-03-14 | E-MTAB-7778 | biostudies-arrayexpress

New insights of Arabidopsis transcriptome complexity revealed by direct sequencing of native RNAs

Project description:The transcriptome profiles of the model plant Arabidopsis thaliana have been extensively studied and charcaterised under different developmental and physiological conditions. However, most of these “RNA-sequencing” datasets have been generated using the sequencing of reverse-transcribed cDNAs from mRNAs that have a relatively short read length. Here, we performed direct RNA sequencing using the latest Oxford Nanopore Technology (ONT) with unusual read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been under-estimated. The ONT direct RNA sequencing technology identified transcript isoforms at a vegetative (14 day old seedlings, stage 1.04) and a reproductive stage (stage 6.00-6) when 10% of the flowers had opened. In-house software called TrackCluster was used to determine alternative transcription initiation (ATI), possible alternative polyadenylation (APA), poly(A) length, alternative splicing (AS), and fusion transcripts. Tombo software was used to detect RNA base modifications. More than 38,500 novel transcript isoforms were identified, including six categories of fusion-transcripts which may result from differential RNA processing mechanisms. Fusion-transcripts are prone to mis-assembly by sequencing with short reads using next-generation-sequencing (NGS). These new transcript isoforms provide important additions to the annotated Arabidopsis genome. The power of ONT in detecting RNA modifications was demonstrated by characterisation of the modifications between mobile mRNAs and total mRNAs. The mobile mRNAs were enriched in m5C modifications, which is consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT direct RNA sequencing greatly enhances the identification of novel RNA transcript isoforms and RNA base modifications.

2020-08-02 | GSE144828 | GEO

Nanopore direct RNA sequencing

Project description:Detection of circRNA through Nanopore direct RNA sequencing

| PRJEB52141 | ENA

Single-cell RNA sequencing of human various osteosarcoma tissues

Project description:Single-cell RNA sequencing of human various osteosarcoma tissues

| PRJNA993663 | ENA

Atlas of RNA sequencing profiles of normal human tissues

Project description:Atlas of RNA sequencing profiles of normal human tissues

| PRJNA494560 | ENA

Direct RNA sequencing of Victoria cruziana

Project description:Direct RNA sequencing of Victoria cruziana