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.