Nanopore DNA sequencing
Nanopore instruments can sequence very long continuous fragments of DNA. Sequencing native DNA allows detection of base modifications.
- good quality high molecular weight DNA (> 30 kb fragment length)
- 100-300 fmol of DNA (depending on fragment length this is equivalent to 1-3 µg of DNA)
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:
- For genome assembly projects it is generally desirable to isolate High Molecular Weight (HMW) DNA.
- DNA molecular weight can be assessed by pulse field gel electrophoresis or instruments such as Fragment Analyzer, TapeStation or Femto Pulse.
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 of DNA can be performed by one of two ways:
- Option 1 – Ligation of Nanopore adapters
- Fragmentation step is optional
- Good preservation of the integrity of the DNA molecule
- Requires more time
- Requires accessible DNA ends
- Very efficient
- Option 2 – Addition of Nanopore adapters by transposon insertion
- Fragments the DNA
- Works with DNA that has inaccessible ends (for example circular DNA)
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 using long fragment application on Fragment analyzer or Femto-Puls.
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.
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.