NGS Library QC: Two Steps, One Instrument

A combination spectrophotometer/fluorometer simplifies nucleic acid concentration and purity measurements.

The Scientist Creative Services Team in collaboration with DeNovix
Mar 23, 2021

T

he success of the library preparation step greatly affects the outcomes of next generation sequencing (NGS) experiments. After researchers isolate nucleic acids, they must perform careful quality control (QC) tests on the resulting material before proceeding with library preparation. Assessing nucleic acid concentration and sample purity is best conducted by two different instruments: a spectrophotometer and a fluorometer.  

Spectrophotometers quickly measure the absorbance of a sample using ultraviolet-visible (UV-Vis) spectrophotometry. This value at a species’ peak absorbance wavelength is proportional to the concentration of that species in the sample. Traditional spectrophotometers use transparent cuvettes to hold liquid samples, but newer microvolume models measure extremely small volumes (0.5-1 µL) on a stage. While spectrophotometers quickly measure absorbance and calculate concentration, they are not very specific for nucleic acid quantification. In the case of DNA library prep, dsDNA, ssDNA, RNA, and free nucleotides absorb at the same peak wavelength of 260 nm, so contaminants increase the measured concentration of the starting sample, resulting in lower amounts of DNA for library prep than expected.

Conversely, absorbance measurements are great for assessing sample purity. Researchers performing NGS experiments must ensure that their nucleic acid samples are free of enzymatic inhibitors, such as proteins, EDTA, phenol, salts, and polysaccharides, which can cause decreased library yield, poor or uneven sequencing coverage, or library preparation failure. Researchers can assess sample purity by analyzing the ratios of 260/280 nm and 260/230 nm measurements because impurities alter the shape of the absorbance spectra if they absorb at similar wavelengths as the molecule of interest.

Fluorometers that measure the emission of fluorescent species are a better choice for assessing nucleic acid concentration prior to NGS library concentration. Because fluorophores bind a target molecule, fluorescence methods are specific and do not measure extraneous nucleic acid species or other contaminants within a sample. Fluorometers are also highly sensitive and can measure sub-picogram amounts of nucleic acid. While these instruments are superior in specificity and sensitivity, fluorescent assays take longer to perform than absorbance readings since scientists must execute additional sample preparation steps and develop standard curves to determine sample concentrations from fluorescent readings.

The DeNovix DS-11 Series of spectrophotometers/fluorometers is the first to combine absorbance and fluorescence readings in their FX models, simplifying QC for NGS applications. The instruments can perform UV-Vis readings on a microvolume scale using SmartPath technology that eliminates sample pipetting errors. Additionally, the FX+ model works with larger sample volumes in cuvettes. These machines can also detect concentrations from fluorescent readings down to 0.5 picogram/µL. The DS-11 Series instruments contain an intuitive touchscreen, pre-programmed assays, and the ability to easily export and share results, enabling researchers to confidently and efficiently prepare NGS libraries.