Quantity and quality of input samples are two key considerations for successful library preparation in next-generation sequencing. High-quality libraries are an important factor to minimize sequencing bias and deliver reliable sequence coverage. A robust quantification and quality control procedure for sample input is therefore essential to ensure consistent results through the workflow and maximize sequencer throughput.
Factors such as specificity, sensitivity, required sample volume, cost and speed are all important factors to consider when selecting a QC method. Understanding the strengths and limitations of fluorometric and absorbance quantitation techniques and using them appropriately will ensure a successful quality control workflow.
Microvolume UV-Vis absorbance
Quantification of nucleic acids by absorbance is performed by analyzing the amount of light transmitted through a sample. The absorbance peak for nucleic acids is at 260 nm, and the amount of light absorbed at this wavelength can be used directly to calculate the concentration of the sample using the Beer-Lambert equation.
Absorbance also provides an indication of sample contamination, as the shape of the absorbance spectrum will change based on the presence of other molecules that absorb at or near the same wavelengths as the molecule of interest.
For genomics and proteomics applications, microvolume spectrophotometers are preferred as measurements are fast, sample volume requirements are minimal (1 µL or less), and the need for sample dilutions is eliminated.
One limitation of using absorbance methods for quantification of nucleic acids is that the peak absorbance at 260 nm is common for all nucleic acid species. Any contaminating ssDNA, RNA, free nucleotides or oligos in a sample of dsDNA for example, will increase the reported concentration. Absorbance methods alone are therefore best suited to the quantification of purified samples.
Fluorescence quantification
Fluorescence methods typically use a fluorophore selective for the biomolecule of interest. When bound to the target, the fluorophore displays enhanced fluorescence allowing specific detection of the molecule of interest.
Fluorescence assays use this binding specificity to establish a direct correlation between the amount of fluorescence emitted by a sample and the concentration of the biomolecule of interest in solution. By mixing a fluorophore with a sample of known concentration and measuring the Relative Fluorescent Units (RFU), a relationship between concentration and measured RFU can be plotted and used as a standard curve. The emission of the same fluorophore, bound to unknown samples, can then be plotted against this standard curve to determine the sample concentration.
The selectivity of fluorescence assays is a key advantage of fluorescence, enabling the researcher to ensure quantitation data is specific for their sample of interest.
Pre-defined apps enable quick and easy measurement of absorbance and fluorescence as well as powerful data export capabilities
Due to the high extinction coefficient of fluorophores, fluorescence assays are also extremely sensitive. Using the DeNovix DS-11 FX and dsDNA quantification assays, for example, concentrations as low as 0.5 pg/µL of dsDNA in the original sample can be detected. This is an improvement in sensitivity of more than 1,000-fold over microvolume absorbance methods.
Most next-gen sequencer providers recommend the quantification of input sample by analyte-specific fluorometric assays such as DeNovix, PicoGreen, or Qubit assays. These methods utilize dyes specific for dsDNA and do not bind to other nucleic acid species that may be present.
Purity assessment
In addition to performing accurate quantification, it is also important to assess the purity of samples to ensure they are free from potential enzymatic inhibitors. Contaminants such as proteins, EDTA, phenol, salts, and polysaccharides can have a negative impact on subsequent enzymatic reactions and lead to decreased yield, uneven or poor coverage, or even library preparation failure. The microvolume UV-Vis absorbance method is recommended for sample quality control. By assessing the ratios of 260/280 nm and 260/230 nm measurements, information on the extent and the nature of impurities in the sample can be inferred.
Summary
Absorbance method advantages
- Quick measurements with no assay costs. The sample is measured directly with no need to set up assays or measure standard curves.
- Wide dynamic range. For the most sensitive microvolume spectrophotometer, the lower detection limit is 0.75 ng/µL of dsDNA (DS-11 Series, DeNovix). By shortening the pathlength (the distance the light travels through the sample) increasingly concentrated samples can be measured. Currently, the upper detection limit for microvolume absorbance is 37,500 ng/µL dsDNA.
- Sample Purity Assessment. Sample quality information can be obtained by analyzing 260/230 and 260/280 ratios.
Fluorescence method advantages
- Specificity. Fluorometric quantification assays bind specifically to the analyte of interest, and results are affected less by common contaminants.
- Sensitivity. Detection limits up to 1,000-fold lower than the microvolume absorbance are possible with fluorescence assays.
Conclusion
Absorbance and fluorescence are distinct but complimentary methods for sample quantification. For NGS labs, combining fluorescence and absorbance methods enables researchers to obtain quantitative and qualitative information for both pre- and post-PCR sample QC while using minimal amounts of sample.
For example, fluorometric quantification confirms if there is a sufficient dsDNA template for a library preparation, and 260/230 nm and 260/280 nm absorbance ratios enable assessment of potentially inhibitive contaminants.
The difference between concentration measurements using absorbance and fluorescence may also give a broad indication of the efficiency of the extraction in isolating only the nucleic acid of interest, as the absorbance value will also include absorbance of all nucleic acids (single nucleotides, oligos, RNA, dsDNA, or ssDNA).
To meet the varying demands of next-generation sequencing labs, DeNovix offers the DS-11 Series of instruments. The DS-11 FX uniquely combines microvolume absorbance and fluorescence measurement modes. Standalone systems for each method are also available in the range and are ideal for labs with specific quantification requirements during pre- and post-PCR procedures.
Andrew Jones is market development manager at DeNovix.
Images: DeNovix
Resources
Video: Guide to Fluorescence Quantification
Related Products from: DeNovix