Nanopore cDNA sequencing
Nanopore cDNA sequencing is able to sequence entire transcripts in one go, ideal for detecting isoforms and fusions events.
Sample requirements
- Good quality RNA with an RNA Integrity Number (RIN) > 7
- total RNA or polyA+ RNA
- concentration requirements vary strongly between applications and input material (1-100 ng)
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
- fragment length analysis using, for example, Bioanalyzer or Fragment Analyzer
- (optional) 260/230 nm and 260/280 nm ratio measured with Nanodrop (does not replace Qubit concentration measurement!)
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
Library preparation
1. PCR-free library preparation
- reverse transcription and strand switching
- generates cDNA from polyA+ RNA
- digestion of RNA template and second strand synthesis
- (optional) ligation of native barcodes
- native barcodes will be used to identify sample identity in a pool when sequenced together on a flowcell
- sequencing adapter ligation
2. Library preparation with PCR amplification
- reverse transcription and strand switching
- generates cDNA from polyA+ or total RNA
- PCR with barcoded primers
- amplification of cDNAs and addition of sample specific barcodes
- 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 cDNA library we might measure DNA concentration and/or fragment length using Bioanalyzer or Fragment Analyzer.
In most cases, the entire library is loaded on the Nanopore flowcell. Each run takes between 24-72 hours depending on the flow cell type.
Expected results
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 provide run statistics and read quality metrics.
Bioinformatics
Last Updated: 24th March 2021