Highly parallel direct RNA sequencing on an array of nanopores

By Daniel R. Garalde, E. Snell, D. Jachimowicz, Botond Sipos, Joseph Lloyd, Mark Bruce, N. Pantic, Tigist Admassu, P. James, Anthony Warland and 16 more. Nature Methods, 2016

1,026 citations recorded by the source corpus.

Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.

Subfields: Cancer Research, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/9ad465668f3c669215305c5adfda48f17512ed8f

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Citations in the corpus

The Architecture of SARS-CoV-2 Transcriptome (2020, positive): “Moreover, because DRS detects RNA instead of cDNA, the RNA modification information can be obtained directly during sequencing (Garalde et al., 2018).”
Diagnosis of plant diseases using the Nanopore sequencing platform (2018, methodology): “Direct RNA sequencing compared with RT-PCR has the advantages that the mean read lengths are long and without PCR bias (Garalde et al., 2018).”
rnaSPAdes: a de novo transcriptome assembler and its application to RNA-Seq data (2018, positive): “Recently emerged long-read protocols for mRNA sequencing allow the capture of full-length transcripts without the assembly [30].”
Piercing the dark matter: bioinformatics of long-range sequencing and mapping (2018, background): “Very recent work has even shown that Oxford Nanopore sequencing can directly read and detect modifications within RNA molecules 115 , something that is not possible to study using second-generation sequencing without using complex protocols.”
The Third Revolution in Sequencing Technology. (2018, neutral): “RNA-Seq Short-read RNA-seq methods are frequently used for gene expression profiling.”
Long-read human genome sequencing and its applications (2020, neutral): “PacBio sequencing technology and ONT sequencing technology are both able to resolve the sequence of full-length RNA molecules, either via cDNA sequencing (PacBio and ONT) 128 131 or via native RNA sequencing (ONT) 122 124 .”
RNA Sequencing Data: Hitchhiker's Guide to Expression Analysis (2018, background): “In contrast to PacBio, ONT also offers direct sequencing of RNA [216].”
DNA sequencing at 40: past, present and future (2017, positive): “Single-molecule methods also open up the intriguing possibility of directly sequencing RNA 66,67 or even proteins 68–71 .”
Transcriptomics technologies (2017, methodology): “Direct sequencing of RNA using nanopore sequencing represents a current state-of-the-art RNA-Seq technique in its infancy (in prerelease beta testing as of 2016) [77][78].”
Accurate detection of m6A RNA modifications in native RNA sequences (2019, methodology): “To overcome these limitations, third-generation sequencing technologies, such as the platforms provided by Oxford Nanopore Technologies (ONT) [18] and Pacific Biosciences (PacBio) ([3], have been proposed as a new means to detect RNA modifications in native RNA sequences.”
SUPPA2: fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions (2018, neutral): “Current technological trends predict an increase in the number of efforts to improve the transcriptome annotation in multiple species and conditions [36].”
Integrative single-cell analysis (2019, background): “Nanopore sequencing holds much promise for multimodal single-cell applications owing to its ability to directly sequence both RNA and DNA with long reads and to natively detect nucleotide base modifications 118–120 .”
Dynamic transcriptomic m6A decoration: writers, erasers, readers and functions in RNA metabolism (2018, background): “Recently, efforts have been made to directly detect m(6)A sites in RNA by engineering DNA polymerase mutant capable of increasing misincorporation opposite m(6)A(147) or by using the nanopore technology for direct detection of RNA modifications.(148) Developing the future direct sequencing technolog”