NGS (Next Generation Sequencing, Next Generation Sequencing) library construction plays a vital role in NGS automated library construction. It is the core link in the entire sequencing process and directly determines the success or failure of subsequent sequencing and the quality of data.
1. Foundation and premise
1. Sample preparation and processing: The first step in automated NGS library construction is usually sample extraction and purification, and library construction is based on these processed high-quality DNA or RNA samples. Without library construction, raw nucleic acid samples cannot be directly used for sequencing.
2. Sequencing platform requirements: Original nucleic acid samples often cannot directly meet the requirements of the sequencing platform, such as fragment length, concentration, etc. The library construction process is a series of processing steps that convert samples into a form suitable for sequencer reading.
2. Core links
1. Fragmentation processing: During library construction, DNA or RNA samples need to be cut into fragment sizes suitable for sequencing. This step is the basis for subsequent sequencing reactions and directly determines the coverage and accuracy of sequencing data.
2. End repair and adapter connection: The fragmented DNA or RNA ends may be uneven and need to be repaired and connected to sequencing adapters. The adapter is the key for the sequencer to identify and start the sequencing reaction, so the success or failure of this step directly determines whether the sequencing reaction can proceed smoothly.
3. Amplification and quality control: In some cases, in order to obtain a sufficient number of DNA fragments for sequencing, PCR amplification may be required. At the same time, library quality control is also a key step to ensure library quality and purity. These operations are completed during the library construction process, providing high-quality libraries for subsequent sequencing.
3. Impact on sequencing results
1. Data quality: The quality of library construction directly affects the quality of sequencing data. If there are problems such as contamination, incomplete adapter connection, or amplification bias during library construction, the accuracy and reliability of sequencing data will decrease.
2. Sequencing efficiency: High-quality libraries can improve sequencing efficiency and reduce errors and waste during the sequencing process. Conversely, poor-quality libraries can result in failed sequencing reactions or poor data quality, increasing experimental costs and time.
4. Advantages of automated database construction
1. Improve efficiency: The NGS automated database construction system can integrate multiple steps, reduce manual intervention and operation time, and improve the efficiency of database construction.
2. Quality assurance: The automated library construction system can improve the quality and reproducibility of library construction by accurately controlling reaction conditions and reducing human errors.
3. Support large-scale sequencing: The automated library construction system can handle a large number of samples, meet the needs of large-scale sequencing, and provide strong support for research in genomics, transcriptomics and other fields.
NGS (Next Generation Sequencing) library construction plays an important role in the NGS automated library construction process and is the key bridge between sample preparation and high-throughput sequencing. It not only requires high-quality DNA or RNA samples as starting materials, but also involves complex molecular biology operations, such as fragmentation processing, end repair, adapter ligation, and possible amplification and quality control steps. Together, these steps ensure that the library meets the requirements of the sequencing platform and provide a solid foundation for subsequent sequencing reactions.
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