For the next-generation sequencing, the quality and concentration of the sample to be tested are two very important factors for the successful construction of the library, and also the key to the success of next-generation sequencing. It minimizes the skew of the sequence and outputs reliable sequence information. Strictly checking the concentration and quality of the samples is the key to ensuring accurate, high-throughput data for next-generation sequencing.
Micro-mode of ultraviolet-visible full-spectrum scanning
The concentration of the quantitative nucleic acid is judged by the absorbance value. The absorption peak of the nucleic acid is at 260 nm, and the absorbance value at this wavelength can be directly calculated by Lambert Beer's law. The absorbance value can also indicate the contamination of the sample, depending on the mixing of different substances in the nucleic acid solution causing a change in the position near the 260 nm in the absorption spectrum.
For genomics and proteomics, ultra-micro-measurement methods are fast-paced, and samples (1 ul or less) are more popular with researchers. It also eliminates the need to dilute the sample.
The absorption peak of the nucleic acid of 260 nm was quantified to determine the sample concentration, but the type of nucleic acid could not be distinguished. For example, ssDNA, RNA, and oligonucleotides, these substances are present as contaminants of the target dsDNA, and directly lead to an increase in absorbance at 260 nm when measured, resulting in an overestimation of the concentration value of the target dsDNA. Therefore, the UV absorption method is only for the purified sample.
Fluorescence quantification method
Fluorescence quantification is achieved by the selective binding of fluorophores to the properties of the target biomolecule. When fluorescence is bound to the target, the intensity of the fluorescence represents the concentration of the target.
Based on this principle, the emitted fluorescence intensity is directly correlated with the concentration of biomolecules in the solution. A standard curve corresponding to the RFU is established by mixing the fluorescent reagent with a sample of known concentration and then measuring its relative fluorescence equivalent (RFU). Then, the sample to be tested is also mixed with the fluorescent reagent, and the detected fluorescent signal RFU is compared with a standard curve to obtain an accurate concentration of the sample to be tested. The specificity of the fluorescent reagent is the key to the fluorescence concentration of the sample. The data obtained by the researcher is guaranteed to be the exact concentration of the target sample.
Fluorescence methods are generally more accurate due to the very high extinction coefficient of the fluorophores. Samples of dsDNA mother liquor down to 0.5 pg/uL can be detected using the DeNovix DS-11FX and dsDNA Quantitative Assay Kit. This is more than 1000 times lower than the lower limit of measurement by the UV absorption method. This method significantly increases the sensitivity of the measurement.
Most of the next-generation sequencing service providers will advise customers to quantify samples sent using fluorescent kits such as DeNovix, PicoGreen, Qubit, etc. The dyes in these kits specifically bind to the dsDNA structure and do not bind to other nucleic acids, thus ensuring that the resulting value is the exact concentration of dsDNA in solution and is not interfered with by other substances.
Sample evaluation purity
An important function to remove precise quantitative concentrations is to eliminate potential enzyme inhibitors such as proteins, EDTA, benzene, salt ions and polysaccharides, which can have a negative impact on subsequent enzymatic reactions. Performance is reduced production, weak coverage, and even library construction failure. Ultra-micro full-spectrum scanning UV-VIS is recommended for monitoring sample purity. By evaluating the values ​​of 260/280 and 260/230, the purity of the sample and the type of impurities mixed can be known.
in conclusion
Although the ultraviolet visible light absorption method and the fluorescence method are completely different, they are all golden rules for sample quantification. For the next-generation sequencing laboratory, combined with fluorescence and UV absorption method, requires only a small amount (1uL), we can ensure that research studies to gain information about the sample concentration and quality. Give follow-up experiments to guide.
In order to meet the research needs of next-generation sequencing laboratories, the new DeNovix DS-11FX/FX+ combines ultra-micro absorption and fluorescence detection modes. The detection capability covers the widest range of sample concentrations available today. Only one instrument needs to be purchased, which meets the needs of all measurement concentrations in scientific research. It has become an essential instrument for next-generation sequencing laboratories.
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