dna quantification needs to be done by fluorescence when nanopore sequencing

this is aimed towards people entering their first year or so of molecular biology work, and i'm sure those with experience already know this. but it begs repeating: you cannot accurately quantify dna using a spectrophotometer, especially when the dna is not pure.

there is a good reason why this needs to be brought up again: many people working with dna will probably be sequencing dna in-house within the next few years with the oxford nanopore platform. a good nanopore sequencing run requires 2 things:

1) pure dna

2) accurate quantification

pure dna

contaminants can partially inhibit the enzymatic reactions required prepare sequencing libraries and damage the pores. pure dna should have a 260/280 ratio around 1.8, and a 260/230 ratio around 2.0 - 2.2. you probably have contaminants if either of these are far off. common contaminants: rna - pure rna has a 260/280 of 2, increasing the 260/280. phenol, edta, guanidine hcl, and carbohydrates - strong absorbance around 230, reducing the 260/230. if you don't have pure dna for nanopore sequencing, throughput and quality will more than likely be negatively affected.

accurate quantification

in addition to requiring pure dna, optimal nanopore results also require a a good estimation of the dna molarity. if you load too many moles of dna, you often get pore blockage/damage, which kills the pores faster, reducing the throughput. if you don't load enough moles of dna, you will have a low pore occupancy, which reduces throughput. you need to get it just right, which means you need to accurately quantify your dna and estimate the molecular weight.

you cannot reliably quantify dna by spectrophotomer, especially if it the dna is not pure. calculating concentration by absorbance ratio is an indirect measurement - any contaminants will negatively affect the estimated concentration. just don't do it. the only questions you can accurately answer with a spectrophotometer (nanodrop, denovix, etc...) are "how pure is this nucleic acid" and "do we have any nucleic acids?".

quantify the dna directly by fluorescence. this can be done with the qubit (quantuas, denovix fluorometer, etc...), and it will result in an accurate quantification.

depending on what the contaminant is, bad dna may also inhibit enzymatic reactions in the library preperation. my guess in this case was edta contamination - this didn't necessarily affect transposase activity in the rapid library prep, but it can signfiicantly affect enzymatic reactions in the ligation sequencing prep. why? because edta will chelate divalent cations (like mg 2+) that are necessary cofactors for some enzymes (like t4 dna ligase). it's always easiest (and cheapest) to start with pure dna rather than troubleshoot if the contamination will affect throughput or not.