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Oxford Nanopore Sample Requirements


Sample Handling

Sample Integrity

Sample Purity

Sample Mass and Concentration Requirements

 

Sample Handling

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.

 

Sample Integrity

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.

 

Sample Purity

DNA should have a 260/280 ratio between 1.8 and 2.0 and a 260/230 ratio between 2.0 and 2.2. RNA samples should have a 260/280 ratio of ~2.0, and the 260/230 ratio should be between 2.0 and 2.2.

Experience indicates that good 260/280 and 260/230 ratios do not guarantee that a DNA sample is truly pure.  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.  The best sequencing results are obtained from DNA samples where the UV-based concentration is less than 150% of the fluorometric-based concentration.

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 5 µg (10 µg preferred) 25 ng/µl
Rapid Barcoding Kit (up to 12 samples) RBK004 Genomic DNA 3 µg 130 ng/µl
Direct RNA Sequencing Kit RNA002 Total RNA 25 µg 500 ng/µl
cDNA-PCR Sequencing Kit PCS109 Total RNA or polyA RNA 1 µg 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 10 µg 50 ng/µl