Oxford Nanopore Technologies
Oxford Nanopore sequencers perform single-molecule long read sequencing of DNA and RNA
Oxford Nanopore have developed a proprietary method for sequencing DNA and RNA through a membrane, detecting base sequence through voltage changes.
Nanopore sequencing uses a helicase to unwind double stranded DNA at a certain speed. Unwound single strands are translocated through biological nanopores as current shifts, corresponding to specific nucleic acid motifs, are recorded in real-time. In addition to DNA Sequencing, nanopore sequencing can also be used for inferring base modifications, such as DNA- and RNA methylation.
To learn more about technology, visit Oxford Nanopore Technologies official site.
NGI offers de novo sequencing using PromethION system
Reference genomes are currently commonly assembled de novo using only long-read technologies because it results in long contiguous assemblies (contig N50 over 1 Mb) with a few scaffolds.
A minimum coverage of 60x is recommended. Some projects might benefit from separate sequencing of a bulk of the data with shorter reads (20-30 kb) that are then enriched with either PacBio or ONT low coverage and ultra-long reads (over 75 kb and up to megabase-size reads if DNA allows).
ONT is a marginally cheaper, yet more challenging technology due to the higher error rate. Its data always requires polishing with Illumina reads (conventional PE or HiC/OmniC). We recommend standalone ONT sequencing only for groups with expertise in bioinformatics. ONT is a good option for projects with a limited amount of DNA, as PacBio requires a much higher input.
Material needed
For specific DNA quality requirements for long-read sequencing technologies see the document (link to DNA quality document).
A bare minimum for 20 kb libraries is 1 ug per flow cell; 2 ug is always preferred. For libraries of 75+ kb insert, at least 5 ug per flow cell are required.
What are pod5 files, and do I need them?
The MinKNOW software on the ONT sequencer records the measurement of the changes in ionic current as a DNA/RNA strand passes through the pore and deposits the information into files in a pod5 file format. These pod5 files are used by a base calling software to determine individual bases, their quality and in some cases also base modifications, which is then written to new files that will be used in downstream processing and analysis.
The base calling software is able to use different models to identify for example different types of modified bases, therefore it can in certain circumstances be advantageous to keep pod5 files, so that base calling can be repeated with a different or updated base calling model. That being said, it does come at a high price: pod5 files can be exceedingly large. Depending on the sequencing yield, the pod5 files of a single PromethION flowcell can have a combined size of over 2 Terrabytes.
In most cases it is not necessary to repeat the base calling and storage costs for pod5 files may quickly exceed the costs of re-sequencing. In fact, later versions of the MinKNOW software will, as a default, not keep the pod5 files after the sequencing run is completed.If you are unsure whether or not your experimental setup requires that you keep pod5 files, please ask a project coordinator before placing an order in the NGI order portal.
Last Updated: 13th May 2026