Nanopore Direct RNA sequencing
Nanopore direct RNA sequencing is able to sequence entire transcripts from native RNA, opening up opportunities to detect RNA modifications.
- 500ng RNA in 9ul nuclease free water (minimal requirement)
- high quality RNA with RNA Integrity Number (RIN) > 7
How to evaluate the sample quality
We check your samples upon arrival, however we still require our users to do their own QC steps before sending samples. For this library prep method, we require:
- Concentration measurements using Qubit
- RNA integrity determination using e.g. Bioanalyzer or Fragment Analyzer
- (optional) ratio of 260/230 nm and 260/280 nm determined by nanodrop or equivalent
If you are not able to carry out these steps, or your samples are below the required thresholds, please get in touch.
What we do with your samples
- Primer annealing and ligation
- adds the primer for reverse transcription and is required to attach the sequencing adapters
- (optional) reverse transcription
- Synthesis of the complementary strand will aid subsequent steps but is not absolutely required. Only the RNA strand will be passed through the pore during sequencing, the cDNA strand will not be sequenced.
- attachment of sequencing adapters
Library QC and sequencing
For most applications using standard library preps we will not perform a formal library QC. Depending on the project and the available DNA we might measure DNA concentration and/or fragment length.
In most cases, the entire library is loaded on the Nanopore flowcell. Each sequencing run takes between 24-72 hours depending on the flow cell type.
The sequencing output is data in .fast5 format which contains the current measurements at the pores. These need to be translated into the base sequence to be useful in downstream applications. Since the base-calling software is in active development, we will on request provide .fast5 files together with the base-called files in .fastq format.
Depending on the project type we will run the nanoseq pipeline (see below), which will not only produce base-called reads but also run statistics and sequence quality metrics.