Sequencing RNA with nanopore technology
How is nanopore sequencing being used in RNA analysis?
- Gene expression studies
- Long reads to study splice variants and fusion proteins
- Identification of sense and antisense transcripts
- Field-based rapid characterisation of RNA viruses and related epidemiology
- Sequencing native RNA allowing RNA modifications to be explored
- PCR-free protocols
- Option for avoiding bias of reverse transcriptases
|cDNA Strand Switching||Direct RNA - Still in Early Access.
These specifications will change
|Workflow||View workflow||View workflow|
|Input amount||50 ng||250 - 500 ng|
|Typical number of full length reads (~1Kb)||Up to 3 million||Up to 1 million|
|Single-molecule accuracy||88 - 91%||84 - 85%|
|Modified base detection||++ (Analysis via open source tools)|
|Poly A tail required||Yes||Yes (or sequence specific with user-supplied primers)|
|Read length||Enriched for full length during PCR||Read length = RNA length|
|Analysis workflow||MinKNOW or Albacore basecalling||MinKNOW or Albacore basecalling|
Nanopore sequencing on the MinION gives you:
Full length transcripts from long reads
Nanopores sequence the whole of the nucleic acid fragment presented to them. For cDNA and Direct RNA sequencing approaches this means full-length transcripts can be sequenced, simplifying the analysis of splice variants, isoforms and fusion transcripts. Methods are available for poly A+ and random priming cDNA libraries for these transcriptomics studies.Publications on full length transcripts
Nanopore sequencing data starts streaming immediately, rather than being delivered in bulk at the end of a 'run'. Real-time data streaming allows rapid identification of sequences within the sample. Depending on the size of the molecule being sequenced, it is possible that the full cDNA transcript will be sequenced in a single read, simplifying mapping to the genome.Publications on real-time analysis
Nanopore sequencing analyses the native nucleic acid
The Direct RNA Sequencing Kit allows RNA to be sequenced directly, without amplification, which can introduce bias. Utilising this kit provides new information on modifications and other properties previously hidden by amplification or reverse transcription stepsPublications on RNA sequencing
Nanopore devices are available at all scales, from portable to benchtop.
The MinION weighs under 100g and can fit in a pocket. It is powered by the USB port on a laptop and is uniquely transportable into the field. With library preparation possible using only a minimum of equipment, field-based analysis labs have been setup in remote destinations. Oxford Nanopore is also preparing to release automated sample and library preparation devices to simplify the workflow even further.
The GridION is a benchtop system integrating real time computing power with the ability to run up to five MinION Flow Cells. The PromethION is a configuration of 48 Flow Cells, each with 6x the capacity of a MinION Flow Cell.Publications on field-based use
cDNA or Direct RNA: which is best for you?
|Applications||PCR cDNA||Direct RNA|
|Splice variants and fusion proteins||***||**|
|Sequencing native RNA allowing RNA modifications to be explored||***|
How are others using the MinION to analyse RNA?
View these publications and movies to find out more.
|02/05/2017||Nanopores allow direct sequencing of full-length RNA strands and modified RNA nucleotides|
|02/05/2017||Multiplexed quantification of protein panels by nanopore sequencing of reporter oligonucleotides|
|02/05/2017||Strand-specific preparation of full-length PCR-based and PCR-free cDNA libraries by strand-switching|
|13/04/2017||Nanopore Long-Read RNAseq Reveals Widespread Transcriptional Variation Among the Surface Receptors of Individual B cells||BioRxiv|
|25/11/2016||The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community||BioMed Central - Genome Biology|
|12/08/2016||Highly parallel direct RNA sequencing on an array of nanopores||bioRxiv|
|03/08/2016||On the study of microbial transcriptomes using second- and third-generation sequencing technologies||Pub Med|
|26/05/2016||Progress at UC Santa Cruz: Long DNA fragments, tRNA and Modified Bases||London Calling event video|
Progress at UC Santa Cruz: Long DNA fragments, tRNA and Modified Bases