Oxford Nanopore Sample Requirements
High molecular weight (HMW) genomic DNA samples must be handled with great care. During isolation and all other handling conditions, HMW DNA must not be vortexed, and any pipetting must use wide bore tips. If wide bore pipette tips have not been purchased, it is acceptable to cut the tip off of a regular pipette tip with a new razor blade. The resulting pipette tip opening should be ~3 mm in diameter. All pipetting actions should be performed slowly. Sample mixing by repeated pipetting will shear the sample. Additionally, the use of low DNA binding microfuge tubes is recommended for HMW DNA.
Non-genomic DNA samples such as amplicons that are less than 20 kbp may be handled according to regular molecular biology practices, but samples that contain longer molecules should be handled with the precautions necessary to prevent physical shearing of the DNA.
RNA samples may be handled according to regular molecular biology practices.
Suggested extraction methods for various organisms are listed on the Nanopore Sequencing Technology page.
A gel image must be submitted along with all DNA samples. The gel must contain a size ladder, which should have an upper marker greater than or equal to 40 kbp (e.g. NEB 1 kb Extended DNA Ladder or Invitrogen 1 Kb DNA Extension Ladder). The gel should be run slowly and sufficiently long so that it will be possible to determine if any sheared DNA exists in the sample. The existence of sheared DNA is an indicator that even the HMW DNA in the sample contains too much damage that will not be corrected by enzymatic nick repair.
DNA must have a 260/280 ratio between 1.8 and 2.0 and a 260/230 ratio between 2.0 and 2.2. RNA samples must have a 260/280 ratio of ~2.0, and the 260/230 ratio must be between 2.0 and 2.2.
Unfortunately, DNA samples can still sequence poorly even if they have good 260/280 and 260/230 ratios. This is true for Pac Bio sequencing as well. A second measure of DNA purity that is often used for long read sequencing is a comparison between the sample concentration as determined by fluorometric (e.g. Qubit) and UV-based (e.g. Nanodrop) assays. If the UV-based concentration is 150% or greater than the fluorometric assay, additional steps should be taken to clean up the sample. If after additional clean up, the UV-based concentration remains more than 150% of the fluorometric assay, the sample will be unlikely to yield a good quality library.
DNA must be treated with RNase to ensure that it is RNA-free.
RNA must be treated with DNase to ensure that it is DNA-free.
Sample Mass and Concentration Requirements
|Library Kit Name||Nanopore Kit ID||Sample Type||Mass Required per Library||Minimum Concentration|
|1D Ligation Sequencing Kit||LSK109||Genomic DNA||2 µg||25 ng/µl|
|1D Ligation Sequencing Kit (without tagmentation)||LSK108||DNA Amplicons||4 µg||90 ng/µl|
|Rapid Barcoding Kit (up to 12 samples)||RBK004||Genomic DNA||1.5 µg||60 ng/µl|
|Direct RNA Sequencing Kit||RNA002||Total RNA||50 µg||500 ng/µl|
|cDNA-PCR Sequencing Kit||PCS109||Total RNA or polyA RNA||500 ng Total RNA or 20 ng polyA RNA||50 ng/µl total RNA or 10 ng/µl polyA RNA|
|Direct cDNA Sequencing Kit||DCS109||Total RNA||20 µg||50 ng/µl|