Today, with the explosion of knowledge and information, how to efficiently integrate, store and retrieve massive data resources has become a major challenge in scientific research and technology applications. Library construction is a key link in information management and data processing. The study of its principles and methods is not only related to the effective organization and utilization of information, but also directly affects the progress of scientific research and the realization of innovation.
Principles of library construction
1. Principles of high-throughput sequencing DNA library construction High-throughput sequencing DNA library construction mainly follows the following principles and methods: 1. Adapter ligation library construction: Basic process: The extracted genome is broken through physical interruption or enzyme digestion. DNA is fragmented. The fragmented DNA ends need to be patched and repaired and A added. Then, adapters are connected under the action of DNA ligase. Finally, the library is constructed through PCR amplification with interspersed purification/sorting steps. Advantages: It can effectively retain almost all genomic information of the sample, which is very conducive to the detection of unknown copy numbers and genomic variations. It is the main library construction method for whole-genome sequencing and exome sequencing. Disadvantages: There are relatively many steps in the library construction process, and multiple magnetic bead purifications are interspersed in the process. The cumbersome steps can easily cause errors. 2. Transposase library construction: Principle: The Tn5 transposon is used to convert multi-step reactions such as DNA fragmentation, end repair, and adapter ligation into one-step reactions, greatly shortening the library construction time. Advantages: The DNA input for library construction is low, and it has a good experience for precious samples or clinical samples with low nucleic acid content; it greatly shortens the library construction time and improves work efficiency. Disadvantages: High requirements for DNA purity and DNA concentration accuracy, otherwise the interruption effect will be affected; compared with traditional ligation adapter library construction, the cost of a single reaction is higher. 3. PCR amplicon library construction: Principle: The PCR reaction is used to add adapters to both ends of the DNA fragment to be tested. The principle is relatively simple. Only two rounds of PCR and two steps of purification are needed to obtain a library of the target region. Advantages: It has clinical applicability and can capture disease target genes, improve the coverage and sequencing depth of target gene detection, and interpret clinical results; it can detect more patient samples through a single sequencing reaction, greatly reducing the task of later biochemical analysis. , the obtained data is easier to store and manage. Disadvantages: When applied to whole-exome or whole-genome library construction, the final library coverage will be low because the designed primers cannot completely amplify all the fragments to be tested; when there is overlap between amplicons, In order to avoid the influence of dominant amplification of short amplicons generated by overlapping portions, two or more primer pools are required, resulting in a complicated experimental process and increased costs.
2. Principles of yeast library construction Yeast library construction is of great significance in proteomics and genomics. Its construction principles mainly include the following methods: 1. SMART technology: Advantages: The initial RNA requirement is very low, and when used as experimental materials When it is difficult to cultivate and the amount of RNA is low, it is more appropriate to choose the SMART method for library construction. During the library construction process, homogenization processing was performed to avoid redundancy of high-abundance genes to a certain extent. Disadvantages: SMART technology requires PCR amplification when constructing a library, and there may be a certain probability of frameshift mismatches, affecting library quality. 2. Gateway technology: Advantages: The library construction process does not require PCR amplification, which can effectively improve the fidelity of the library. At the same time, a primary library will be generated, which can theoretically be used multiple times and constructed into other destination vectors. Under normal circumstances, Gateway technology can meet most library construction needs. Disadvantages: Gateway technology will undergo two recombinations when constructing a library. The shaking amplification process of the primary library is also similar to the PCR process. It will also cause redundancy of high-abundance genes and loss of low-abundance genes, affecting the quality of the library. 3. In-Gate technology: Principle: In-Gate technology improves SMART technology and Gateway technology, and has the advantages of Gateway technology without PCR amplification and high fidelity. At the same time, only one step of recombination is performed to reduce the loss of low-abundance genes and the redundancy of high-abundance genes, resulting in a higher full-length library rate and positive rate. Advantages: Compared with other traditional technologies, In-Gate technology has obvious advantages in library quality. Disadvantages: As a new technology, its application scope is not wide yet.
The principles of library construction involve multiple fields and complex technical paths, but whether it is a DNA library, a protein library, or other types of libraries, the construction process is designed to achieve effective integration, storage, and retrieval of information. By in-depth understanding of the basic principles of library construction, we can better select and apply suitable technical methods, optimize the library construction process, and improve the quality and efficiency of the library.
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