Nanopore - Whole Genome Sequencing
Nanopore Whole Genome Sequencing offers two different protocols, the Rapid Protocol and the Ligation Protocol. The Rapid Protocol is based on transposase mediated fragmentation and optimized for fast and easy sample processing, lowering the cost of sample processing compared to the ligation protocol. However, due to missing clean-ups and fragmentation of input DNA, output and read length is lower as well. In the ligation protocol, the adapters are ligated to the DNA and fragmentation is optional. Therefore, the output and read length are larger (if no fragmentation was done). Up to 24 samples can be multiplexed.
The output of a Flow Cell depends significantly on the quality, quantity and fragment length of the source material. It usually varies between 5 and 15 Gb. In general, longer fragments will lead to longer reads but will also decrease the overall yield, while shorter DNA-fragments will lead to shorter reads but higher overall yields. Since we need about 200 fmol gDNA for the ligation, the amount of DNA required varies depending on the fragment length of the gDNA.
The total cost depends significantly on the required coverage. Here is a small overview of rough guidelines regarding the necessary coverage. for a project specific quote.
- Gap Filling: The Nanopore Assembly is only used to bridge gaps or ambiguities in an already existing short-read scaffold genome
- Structural Variants (SV) Detection: Here, the de novo assembled Nanopore genome is mapped against a known reference genome to detect large structural differences (e.g., insertions / deletions / inversions)
- Hybrid assembly: Here, long and short read data are combined in one assembly in order to minimize the errors of both approaches
- De novo assembly: A completely new, high-quality genome of the organism is created, ideally with one sequence per chromosome / plasmid