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Next-generation sequencing (NGS), also known as massively parallel sequencing, can simultaneously sequence millions or even billions of DNA molecules, achieving the goal of large-scale, high-throughput sequencing. This revolutionary advancement, following Sanger sequencing, is of groundbreaking significance. Because high-throughput sequencing can detect a large number of target genes and their variant sites simultaneously, it offers high sensitivity and specificity, combining both qualitative and quantitative detection. Compared to other methods, it is relatively inexpensive for testing the same number of genes and sites. Therefore, it has broad clinical and scientific application prospects in areas such as non-invasive prenatal screening (NIPT), tumor gene mutations, genetic diseases, preimplantation genetic screening (PGS), preimplantation genetic diagnosis (PGD), pathogenic microorganisms, and metagenomics. It has become the most efficient tool for DNA and RNA sequence analysis and a supporting technology for research and clinical diagnosis and treatment in the era of precision medicine. It has enabled genomics and transcriptomics to rapidly advance from conceptualization to clinical practice, heralding the arrival of a molecular era in disease diagnosis and treatment. Currently, in clinical oncology practice, NGS is primarily used for driver gene sequencing, a crucial component of precision cancer diagnosis and treatment. Characteristics of high-throughput sequencing technology: The operation steps are numerous and complex, including sample pretreatment, nucleic acid extraction, quantification, quality control and fragmentation (for genomic or large-fragment nucleic acid molecule detection), library construction, amplification, target sequence enrichment, library quantification, sample mixing, sequencing preparation, and sequencing. >Subsequent bioinformatics analysis processes also include post-sequencing data quality analysis, alignment, variant identification, annotation, and result reporting and interpretation. Problems in any of the above links will affect the accuracy of the test results and thus affect clinical decisions. The problem exists that the value is lost: Currently, the vast number of cancer patients in my country is driving a significant demand for genetic diagnosis and testing applications. However, due to the low barrier to entry for testing technology and the lack of clinically recognized NGS guidelines, cancer diagnosis and treatment can be plagued by poor testing quality, inaccurate results, unprofessional interpretation of test results, and even blind pursuit of genetic testing results. Consequently, if problems arise in these areas, research and diagnosis will lose their value. Fortunately: In July of this year, the Chinese Medical Journal published an online article titled "Expert Consensus on the Application of Next-Generation Sequencing Technology in Precision Medicine Diagnosis of Cancer." The article systematically expounds on the quality requirements of NGS technology, clinical oncology-related NGS testing content, sample processing, sequencing workflows, data management, informatics analysis, result report interpretation, and consultation. It also discusses and provides regulatory recommendations on various issues in the clinical application of NGS. See here for scientific knowledge: Solve existing problems Enhance existing value Double the efficiency | Achieve tumor precision Quality Control of Nucleic Acid Samples for Next-Generation Sequencing Provide you with full process solutions 1 Sample Processing in NGS Applications NGS testing requires library construction, and the raw materials for this step are DNA or cDNA derived from RNA. Sample types suitable for clinical NGS analysis are preferably fresh tissue specimens, but formaldehyde-fixed and paraffin-embedded (FFPE) specimens, tumor cytology specimens, and plasma (free DNA/RNA) can also be used. Collection of various samples: (1) For fresh tissues from surgery and biopsy: The ideal preservation method is to quickly place them in liquid nitrogen, which can be stored in a liquid nitrogen tank or a -80°C freezer. This process should be completed within 30 minutes after surgery to prevent degradation of nucleic acids such as RNA; or they can be stored in a sample protectant and transferred to a -80°C freezer as soon as possible. Frozen section staining can be used to assess the tumor cell content in the sample. (2) FFPE: Samples should be obtained according to pathological operation specifications. Tissues removed by surgery or biopsy should be immersed in 100 ml of 4% formaldehyde solution for fixation within 30 minutes. Avoid using acidic fixatives or those containing heavy metal ions. Large specimens should be fully fixed for 6 to 48 hours after dissection, but not more than 72 hours. Small biopsy specimens can be fixed for 6 to 12 hours. HE staining should be performed to assess the content of tumor cells before NGS testing. (3) Cytology samples: When exfoliative cytology specimens and fine needle aspiration cytology specimens are used for genetic testing, pathological quality control must be performed to determine the number of tumor cells in the specimen and the ratio to normal cells. Specimens that meet the quality requirements or meet the requirements after tumor cell enrichment can be directly extracted for nucleic acid or prepared into FFPE cytology wax blocks for subsequent analysis. Circulating tumor DNA (ctDNA) can be extracted from the cell-free supernatant of body cavity effusion specimens for testing. (4) Plasma or blood samples: ctDNA is free DNA present in plasma. The proportion of tumor-derived DNA in plasma free DNA varies greatly among different tumors and cases. When collecting peripheral blood to extract plasma free DNA for testing, a disposable anticoagulant vacuum blood collection tube containing ethylenediaminetetraacetic acid (EDTA) should be used. 8-10 ml of whole blood should be collected and transported refrigerated. Plasma should be separated within 2 hours, free DNA should be extracted, and stored in a -80 ℃ refrigerator to avoid repeated freezing and thawing. If peripheral blood needs to be transported for a long time, it is recommended to use a commercial free DNA sample storage tube (Qihengxing cfDNA blood collection tube is recommended, which is high quality and cost-effective). Under room temperature conditions, ctDNA can be stably stored in whole blood for up to 14 days (Qihengxing). Since the potential large-scale release of blood cell genomic DNA will greatly dilute the concentration of plasma free tumor DNA, samples that are confirmed to be hemolyzed by naked eye observation are not suitable for NGS detection of free tumor DNA. When it is suspected that plasma free DNA is contaminated by blood cell genomic DNA, nucleic acid fragment size distribution analysis can be considered to determine whether contamination exists and whether the sample is suitable for NGS testing. 2. Nucleic acid extraction Whether it is a blood sample, fresh tissue, or FFPE sample, the products on the market are relatively mature (such as Qiagen, Life, Promega, etc.). Choose a membrane column method or magnetic bead method nucleic acid extraction kit based on the sample size and specific needs. 3 Nucleic acid quality assessment First, it is essential to evaluate the quality (integrity and purity) of genomic DNA and RNA. However, for cfDNA, the concentration obtained after extraction is typically very low, and spectrophotometry and gel electrophoresis are not typically used for quantification and purity analysis. However, dye-based methods (such as qubits) are used for nucleic acid quantification, and microfluidics methods are used to analyze fragment size and the presence of genomic contamination. Gel electrophoresis The integrity and size of genomic DNA can be assessed using conventional or pulsed-field agarose gel electrophoresis (PFGE). While conventional gel electrophoresis is less precise, it still provides valuable information on integrity (size range) and purity (RNA contaminants forming tailed bands at the bottom of the gel). Therefore, it remains a valuable method for assessing genomic DNA and RNA quality. Note: RNA contamination can lead to an overestimation of DNA concentration and inhibit some downstream steps. If RNA contamination is suspected, treat the sample with DNase-free RNase I. Spectrophotometry: The principle of spectrophotometry is that nucleic acids, nucleotides, and their derivatives all possess conjugated double bonds that absorb ultraviolet light. The UV absorption peaks for RNA and DNA are at 260 nm. Generally, at 260 nm, the absorbance of a solution containing 1 μg of RNA per ml is 0.022–0.024 nm, and the absorbance of a solution containing 1 μg of DNA per ml is approximately 0.020 nm. Therefore, measuring the absorbance at 260 nm of an unknown RNA or DNA solution can be used to calculate the nucleic acid content. This method is simple and rapid. However, if the sample contains a large amount of UV-absorbing substances such as nucleotides or proteins, the photometric error will be significant, so these substances should be removed beforehand. NanoDrop and other UV spectrophotometers use UV absorbance for detection, which cannot distinguish between DNA, RNA, degraded nucleic acids, free nucleotides, and other impurities. Microfluidic analysis Microfluidic analysis (such as the PE LabChip GX and Agilent 2100) utilizes microfluidics to separate samples. By applying voltage, samples bound to fluorescent dyes are separated within microscopically etched channels on the chip. Separation is achieved based on the size and mobility of nucleic acids. Excitation light causes the dyes to fluoresce, enabling detection by the instrument. Using a ladder with known molecular weight and concentration, the relationship between DNA migration time and size is calculated, and the sample's molecular weight and concentration are then calculated using a formula. The second step is to determine the concentration of genomic DNA. Spectrophotometry Nucleic acid concentration can be determined by measuring absorbance at 260 nm using a spectrophotometer. Molecular Devices' Quickdrop and Thermo Fisher Scientific's Nanodrop are widely used because they require small sample volumes (0.5-1 µl) and are easy to use (no cuvettes are required). To ensure reliable results, readings should be between 0.1 and 1.0. Note: Absorbance measurements cannot distinguish between DNA and RNA. RNA contamination can lead to an overestimation of DNA concentration. However, the A260/A280 ratio of pure RNA is approximately 2.0, while that of pure DNA is approximately 1.8. Therefore, a value of 1.95 indicates RNA contamination in the sample. Note: Phenol has a maximum absorbance in the 270–275 nm wavelength range, very close to that of DNA. Therefore, phenol can increase the sample's absorbance around 260 nm, leading to an overestimation of DNA yield and purity. Fluorescence methods use fluorescent dyes to determine DNA concentration, offering specificity and sensitivity. Binding of the dye to DNA/RNA increases the intensity of luminescence at specific wavelengths. Alternatively, more sensitive fluorescent dyes, such as PicoGreen, can be used. PicoGreen-based assays are 10,000 times more sensitive than UV spectrophotometry and at least 400 times more sensitive than methods using Hoechst 33258. Unlike UV spectrophotometry, PicoGreen assays are much more selective for double-stranded DNA than for RNA and single-stranded DNA. DNA standards and samples are mixed with a fluorescent dye and detected using a fluorometer. The sample results are compared with those of the standards to determine the DNA concentration. Library preparation For the Illumina sequencing platform, a typical DNA library construction protocol consists of four steps: Fragment DNA (KAPA enzyme digestion kit is optional); Perform end repair on DNA fragments; Connecting joints; Amplify the optional library. I won’t go into the details of the database building process here. If you are interested, you can read our previous content for a very detailed introduction. NGS library quality control. High-quality libraries are crucial for successful next-generation sequencing. Library construction involves complex steps, such as fragmenting the sample, repairing the ends, adenylylating the ends, ligating adapters, and amplifying the library. These steps vary depending on the platform and library type used. Monitoring each step is essential, including checking the size of the fragments after fragmenting the sample and checking the size and concentration of the fragments after ligating adapters. During library validation, the size and quantity of the fragments in the library are analyzed as the final step in quality control. Estimating the size of fragments in the library Agarose and PAGE gel electrophoresis are traditional methods for detecting fragment size, which are time-consuming. Recently, microfluidic electrophoresis or capillary electrophoresis has become increasingly popular for fragment size and concentration analysis. Ready-to-use chips and cartridges eliminate the need for gel preparation, making them easy to use. They offer higher throughput and significantly reduce hands-on time. Furthermore, they offer higher sensitivity (limited detection) and fully automated data acquisition and electronic output. These instruments can simultaneously measure both fragment size and concentration. Electrophoresis and capillary electrophoresis based on microfluidics (LabChip, 2100) provide quantitative detection data in addition to fragment size information. However, a common limitation of electrophoresis, spectrophotometry, and fluorescence is that they only measure the concentration of total nucleotides, not the concentration of molecules attached to the adapter. Real-time PCR Accurately quantifying the number of molecules in the amplified library is crucial for ensuring fragment quality and efficient data acquisition. Underestimating the number of molecules in the amplified library results in mixed signals and difficult-to-interpret data; conversely, overestimating the number of molecules reduces the yield of beads or DNA clusters bound to the template and underutilizes sequencing capacity. Real-time PCR can specifically and quantitate DNA molecules with adapters attached to both ends, enabling highly accurate quantification of molecules in an amplified library. Real-time PCR's exceptional sensitivity allows for quantification of library molecules at very low concentrations, even below the detection threshold of traditional methods. This method therefore minimizes library amplification and reduces potential bias. |
