{"id":8513,"date":"2025-01-16T17:40:50","date_gmt":"2025-01-16T09:40:50","guid":{"rendered":"https:\/\/opentrons.com.cn\/news\/\/"},"modified":"2025-01-16T17:41:46","modified_gmt":"2025-01-16T09:41:46","slug":"wenkudingliangdefangfa","status":"publish","type":"post","link":"https:\/\/en.opentrons.com.cn\/news\/wenkudingliangdefangfa\/","title":{"rendered":"Common methods and techniques for library quantification"},"content":{"rendered":"\n

In the vast field of exploring high-throughput sequencing (NGS), library quantification plays a crucial role as a key link in ensuring the quality and depth of sequencing data. This step not only concerns the success of the experiment, but also directly affects the accuracy and reliability of subsequent bioinformatics analysis. With the continuous advancement of technology, the methods of library quantification are becoming increasingly diverse. Each method has its own characteristics and is suitable for different experimental scenarios and needs.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Library Quantification<\/a><\/p>\n\n\n\n

1. The importance of library quantification 1. Ensure the quality of sequencing data: Library quantification can ensure that the amount of DNA libraries loaded on the sequencer is moderate, thereby avoiding insufficient sequencing data due to excessive library concentration or insufficient sequencing data due to excessive library concentration. 2. Stabilize the output of each sample data: When multiple libraries are combined and sequenced, library quantification helps to mix corresponding molar amounts of libraries according to the requirements of the sequencing data volume to stabilize the output of each sample data volume.<\/p>\n\n\n\n

2. Common methods 1. UV spectrophotometry (1) Principle: The bases in nucleic acids (including DNA and RNA) contain aromatic ring structures, so they have the characteristics of ultraviolet absorption. The ultraviolet absorption peak of nucleic acid is at 260 nm. At the same time, the purity of the nucleic acid can be estimated by calculating the values \u200b\u200bof A260\/A280 and A260\/A230 (280 nm light absorption usually comes from proteins, while 230 nm usually comes from sugars and phenol, etc.). (2) Disadvantages: It is impossible to distinguish between DNA and RNA, because the structure with ultraviolet absorption is the base of nucleic acid, while both DNA and RNA contain bases. Therefore, when a sample contains both DNA and RNA, the concentration of the determination will be higher than its true concentration. 2. Fluorescent dye method (1) Principle: Use target-specific dyes, which can specifically bind to different types of nucleic acid strands and emit fluorescence under the excitation of light sources at a specific wavelength. Among them, the signal can increase by dozens to hundreds of times compared to those free, so it can be distinguished from the background. (2) Operation: Mix the DNA sample to be tested with the fluorescent dye, and the fluorescent dye will bind to the DNA to form a fluorescent complex. The mixture is then placed into a Qubit measuring instrument, which excites the fluorescent complex by laser, causing it to emit a fluorescent signal. The Qubit measuring instrument will automatically calculate the DNA concentration based on the intensity of the fluorescence signal. (3) Advantages: High sensitivity, easy and fast operation, efficient detection process, and can easily analyze dozens of samples. The built-in analysis software simplifies data calculation and presents the library concentration immediately after the detection is completed. (4) Disadvantages: When the samples increase to more than 20~30, this detection method is not very suitable; the detection accuracy is slightly lower, and the result is generally not used as the final concentration for on-machine. 3. Real-time quantitative PCR method (qPCR) (1) Principle: Use fluorescence detection technology and PCR technology to add fluorescence groups to the PCR reaction system, and monitor the changes in fluorescence signals during the entire PCR process in real time to reflect the changes in concentration. Finally, the concentration of unknown samples is calculated through the standard curve of known concentrations to achieve accurate quantitative library concentration. (2) Advantages: Specific primers can only quantify the complete library of two end joints, which can eliminate interference from unsequencing libraries that do not connect to the linker with single-ended or double-ended joints. It has high accuracy and is currently recommended for library quantification in the industry. It is suitable for measuring precious samples and accurate quantification before library pooling. (3) Disadvantages: High cost.<\/p>\n\n\n\n

3. Skills 1. Eliminate and avoid nucleic acid pollution: The concentration of Standards used in the library quantitatively ranges from high to low. High concentrations of Standards are likely to cause nucleic acid pollution to the environment, thereby affecting the results of negative controls and low concentrations of Standards. Therefore, laboratories that do library quantification need to remove nucleic acids regularly. After the experimental technique, nucleic acids in the environment can be oxidized with ultraviolet treatment. 2. Library dilution verification: The library recommends diluting 2 gradients and verifying each other. The instructions are recommended to be 10,000 times and 20,000 times. It is also possible to set two gradient concentrations to 10 times during specific experimental operations. It should be noted that the difference between the Ct values \u200b\u200bobtained by 10,000 times and 20,000 times should be around 1, which can be between 0.951.05, and the difference between the Ct values \u200b\u200bobtained by 10,000 times and 100,000 times is around 3.34, which can be between 3.23.5. If these two intervals are exceeded, the error of the two gradients will be relatively large, which may be caused by inaccurate dilution. 3. Exclude the impact of amplification efficiency: When the calculation results are found that the concentration deviation obtained by diluting the library into two gradients is large, it may be due to inaccurate dilution, or the amplification efficiency of the library is low, resulting in large \u25b3Ct. At this time, this library can be made into 4 to 5 10-fold dilution gradients, and the amplification efficiency can be calculated based on the Ct value of the onboard, thereby eliminating the factors of amplification efficiency.<\/p>\n\n\n\n

Library quantification is a key link in the high-throughput sequencing process, and its importance is self-evident. By rationally selecting quantitative methods and flexibly applying quantitative techniques, scientific researchers can more accurately grasp the quality information of the library, laying a solid foundation for subsequent bioinformatics analysis and scientific research.<\/p>\n\n\n\n

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<\/p>\n","protected":false},"excerpt":{"rendered":"

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