What are the methods used for quantitating next generation sequencing (NGS) libraries?

There are several methods for quantitating next generation sequencing (NGS) libraries. They vary widely in terms of ease and accuracy. The four main methods for quantifying NGS libraries are qPCR, fluorometry, spectrophotometry, and electrophoresis

• Quantitative PCR (qPCR) offers the ultimate accuracy in quantitation. It quantitates library preps against standard curves using adaptor-specific primers with fluorescent dyes and primers. Quantitating only these viable sequencing templates offers the best chance at normalizing DNA libraries accurately. This is a very sensitive method of quantifying NGS libraries and is the preferred option for quantitating rare sequences and libraries of lower concentration. The downside of this method is it is more hands-on than other methods and the reagent costs can start to add up. 
• Fluorometry assesses the concentration of nucleic acids against a standard curve using dsDNA-specific intercalating fluorescent dyes that bind specifically with high affinity to DNA or RNA. This method is reasonably fast and low cost, and offers sensitive and accurate estimation of concentration of dsDNA because only target-bound dye emits a fluorescent signal. It can also be used to specifically quantitate protein, single-stranded DNA, and RNA. The downsides of fluorometry are the inability to differentiate between adaptor-ligated and other molecules or the inability to estimate fragment size. 
• Electrophoresis estimates sizes of DNA fragments through capillary electrophoresis and concentration through intercalating dyes. It is the preferred method of estimating fragment size and distribution because of its high level of accuracy. The downside of this method is it is unable to distinguish between adaptor-ligated and other molecules. In addition, the equipment requirements are expensive considering they can only be used for a single purpose. 
• Spectrophotometry is a quick and low cost method for quantitating NGS libraries. It detects the absorption of UV light by molecules in the sample and calculates the concentration against a standard curve. The estimated purity is based on the ratio of absorbance measured at 260 and 280 nm. The downsides of this method are low sensitivity and inaccuracy as it quantitates all nucleic acids, not just adaptor-ligated molecules. In addition, the results may be affected by contaminating proteins and RNA.