Quantification and Quality Control: Critical Considerations for cfDNA Analysis in NGS Applications
Accurate cfDNA quantification is essential for successful NGS workflow. This article discusses the enhancement of sample quality control using the high sensitivity and specificity of the EzCube Fluorometer. It offers practical guidelines for integrating the EzCube and EzDrop Micro-Volume Spectrophotometer into cfDNA pre-processing and sequencing preparation.
Cell-free DNA (cfDNA) has demonstrated significant promise in next-generation sequencing (NGS), especially in the field of precision medicine for cancer. Its real-time monitoring and non-invasive characteristics make it an important tool for tumor molecular analysis. cfDNA can overcome the problem of tumor heterogeneity and provide more comprehensive tumor genomic information than traditional tissue biopsy. It greatly improves the detection rate of actionable genetic variants and is an alternative option in cases where tissue samples are scarce or difficult to obtain.
However, cfDNA analytical procedures face many challenges, such as an extremely low proportion of tumor DNA (ctDNA) in the total cfDNA (0.01% to 10%). On a technical level, cfDNA sample concentration can be extremely low with a high contamination risk. To address these challenges, studies have emphasized the need for rigorous quantification and quality control (QC) to support broader adoption in clinical practice.
Why does cfDNA NGS require higher standards of quantification and QC?
When conducting NGS analysis of cfDNA, strict quantification and QC standards are essential, mainly because of the special properties and technical challenges of cfDNA itself. The content of cfDNA in plasma is extremely low (usually 2-10 ng/mL), and the short fragments are easily degraded. If the sample quality is not accurately controlled in the NGS process, the following problems may occur:
- Library construction failure: The library construction stage may fail due to insufficient cfDNA, making subsequent PCR amplification impossible.
- Quantitative error: Inaccurate quantification will lead to deviations in the estimation of library concentration. Given that cfDNA exhibits extremely low concentration (<1 ng/μL) amidst abundant background DNA, such inaccuracies can cause excess, or insufficient coverage during sequencing.
- Data deviation: The ~166bp fragment profile is critical for quality assessment. The presence of high-molecular-weight genomic DNA (gDNA >800bp) or other impurities in the sample will seriously affect the sequence alignment (mapping) efficiency and repetition rate and cause data deviations.
To overcome these hurdles, the NGS processing of the cfDNA must have very high sensitivity and reproducibility. It must accurately quantify extremely small amounts of nucleic acids, and must also be able to detect sample degradation and contamination to ensure that the sequencing data is of the highest quality. Although traditional spectropho-tometry is simple and fast and can provide preliminary assessment of total concentration and purity of a sample, its sensitivity and specificity are often inadequate when faced with low-concentration samples such as cfDNA that are very sensitive to impurities.
Therefore, in research and clinical laboratories, there is a growing trend towards more sophisticated detection methods, especially when used with a highly sensitive and accurate fluorometer (such as the EzCube. This type of instrument can not only accurately quantify extremely low concentrations of cfDNA, but also effectively evaluate sample integrity, provide reliable quality control for subsequent NGS library construction and sequencing, and ensure the accuracy and reproducibility of experimental results.
How the EzCube Fluorometer Supports Accurate cfDNA Handling
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Higher sensitivity and accuracy
The Fluorometer uses fluorescent dyes to specifically bind to DNA/RNA and can handle samples with extremely low concentration. It shows highly accurate and repeatable results in samples with concentrations of a few picograms per milliliter. It is particularly suitable for sample quality testing in NGS experiments.
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Specific detection of DNA/RNA
As mentioned above, if the sample pretreatment involves the use of more complex reagents, it may also interfere with the detection reaction. However, the fluorescent dye used in the fluorimetry only binds to DNA or RNA and the results are not affected by the presence of proteins, salts or other contaminants. Therefore, it can more accurately reflect the actual concentration of DNA/RNA in the sample. This specificity supports more reliable quantification in NGS sample quality control.
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Easy and fast operation
The fluorometer is user-friendly and easy to operate. Just mix the sample with the fluorescent dye and put it into the instrument. Results are obtained within a few seconds. These results are very accurate and reliable even with very low-concentration samples. The efficiency and convenience of the fluorometer make it a practical choice for testing sample quality in NGS workflows.
Dual Quality Control: The Gold Combination of EzCube and EzDrop
During sample pre-processing, reliance on a single instrument may not always be enough to ensure perfect and rapid assessment of both sample concentration and purity. However, combination of the EzDrop with the EzCube offers complete and reliable support for downstream results:
- EzDrop Spectrophotometer: A fast, simple, and economical nucleic acid/protein quantification tool that can detect high-concentration samples (eg, dsDNA 2–20,000 ng/μL) without reagents. It is suitable for preliminary quantification and purity analysis, and can detect contamination which is a key to sample quality control (see Table below).
- EzCube Fluorometer: Ultra-high sensitivity (minimum 0.01 ng/μL) and specificity, can distinguish nucleic acid types (dsDNA/ssDNA/RNA). It requires fluorescent reagents, but is designed for trace detection and high precision experiments (such as NGS/qPCR).
Table: The combination of the EzDrop and the EzCube provides a complete sample preparation solution
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Features for QC |
EzDrop Spectrophotometer |
EzCube Fluorometer |
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Quick concentration check |
★ Instant, no reagents required |
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Accurate dsDNA quantification |
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★ High specificity |
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Purity assessment (A260/280 & A260/230) |
★ Detects contaminants (eg, protein, solvent) |
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Low-concentration detection |
- Limited reliability (high variability) |
★ Highly sensitive (low detection limit) |
The combination of the EzDrop and the EzCube provides a complete sample preparation solution. They can measure the total amount, purity, and effective concentration of samples, help reduce NGS failure rates, and are an ideal combination in the cfDNA pre-processing workflow.
Related Post:
Spectrophotometers vs. Fluorometers: Choosing the Right Tool for DNA/RNA Quantification
Experimental Recommendation: Application Guidelines for cfDNA Liquid Biopsy Workflow
If you are conducting cfDNA-related NGS research, we recommend that you use the following sample quality control process to improve the experimental success rate and data quality:
- Using an EzDrop spectrophotometer, the absorbance of the sample at 260nm should be measured to assess the nucleic acid concentration, and the A260/280 and A260/230 purity ratios should also be measured to ensure the sample quality meets the requirements for subsequent experiments.
- Using an EzCube fluorometer, the dsDNA concentration can be accurately quantified by combining different types of EzQuant reagents to ensure that the sample reaches the starting amount required for library construction. This method has pg-level sensitivity and can effectively distinguish the concentration differences between samples indicating malignancy and normal ones.
- After library construction, the fragment distribution should be analyzed using a capillary electrophoresis system, and the effective concentrations can be verified by qPCR if necessary to ensure sequencing quality.
This recommended workflow can be applied across various cfDNA study settings, providing a structured approach to pre-sequencing quality control.
Accurate quantification lays the foundation for NGS quality
cfDNA samples contain rich biomarker potential, but analysis is a challenge. To obtain reliable next-generation sequencing results, rigorous quantitative tools and quality control are essential. The EzCube fluorometer quantifier has become a key tool for low-concentration cfDNA research with its pg-level detection sensitivity and excellent specificity. Support from the EzDrop spectrophotometer allows quick evaluation of the purity and total amount of samples. The two complement each other to build a complete quality control mechanism.
In both basic research and clinical testing, strict control at the sample pre-processing stage is vital to ensure the reliability of subsequent NGS data. By combining accurate quantification with robust purity assessment, this approach not only improves reproducibility, but also reduces library preparation failures and minimizes sequencing costs.
Related Post:
Comprehensive Nucleic Acid Quality Control-Total Solutions for Molecular Experiment Issues
References
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- Blue-Ray Biotech. (n.d.). EzCube Fluorometer. Retrieved from https://www.blue-raybio.com/en/category/product/EzCube-Fluorometer
- Blue-Ray Biotech. (n.d.). EzQuant Quantification Assay Kits. Retrieved from https://www.blue-raybio.com/en/category/product/EzQuant-Quantification-Assay-Kits