With the rapid development of technology today, NGS (Next-Generation Sequencing) technology has occupied an important position in biomedical research due to its high throughput, high efficiency and high accuracy. As a cutting-edge gene sequencing technology, NGS has not only greatly promoted the development of genomics, transcriptomics and other fields, but also provided strong support for the diagnosis, treatment and prevention of diseases.
1. DNA extraction and quality inspection1. Sample preparation: Obtain pure, sufficient and good-quality sample DNA from various sources such as plasma and fresh tissue. 2. DNA extraction: Use appropriate extraction methods, such as phenol chloroform extraction method, magnetic bead method, etc. to extract DNA in the sample. 3. Quality inspection: The concentration, purity and integrity of DNA are detected by electrophoresis, spectrophotometers, etc. to ensure that the DNA quality meets the requirements of subsequent experimental results.
2. Library construction1. DNA processing: break the DNA into a length interval suitable for sequencer reading (such as 200-300bp), and perform terminal repair, fragment screening and other treatments. 2. Add linker: Add specific linker sequences to both ends of the DNA fragment. These linker sequences contain binding sites for sequencing primers for subsequent sequencing reactions. 3. PCR amplification: Using the linker sequence as a template, a sufficient number of DNA libraries were obtained by PCR amplification.
3. Targeted Capture (optional)For sequencing projects in specific regions (such as whole exome sequencing, genome-specific region sequencing, etc.), targeted capture technology is required to efficiently enrich the target sequence from the library. This step can improve the accuracy and depth of sequencing data, helping to more targeted analysis of genes or regions of interest.
IV. On-machine sequencing1. Library loading: Load the prepared library into the flow cell of the sequencer to provide the necessary samples for subsequent sequencing. 2. Sequencing reaction: The sequencer uses the method of synthesis, sequencing or ligation and sequencing to sequence the nucleic acid fragments in the library. During the sequencing process, the sequencer will gradually introduce fluorescently labeled dNTP (deoxyribonucleoside triphosphate) and determine the base type at each position by monitoring the fluorescence signal. 3. Image capture and data analysis: Use a high-resolution camera to capture the image of fluorescent signals, and then process and analyze the image and sequencing data through bioinformatics software to obtain complete sequencing results.
5. Data Analysis1. Primary Analysis: Convert light intensity data into sequence information (AGCT), that is, the original sequencing data. 2. Secondary analysis: It is mostly automatically completed by the instrument, including base quality scoring, sequence alignment, variation detection and other steps. 3. Third-level analysis: It involves the processing of vcf files, and can be personalized adjustments, such as gene annotation, variant filtering, etc., to obtain the final sequencing results and biological significance.
6. Results Interpretation and Report Based on the results of data analysis, interpret the variation information in the sequencing data, such as single nucleotide polymorphism (SNP), insertion or deletion (InDel), structural variation, etc., and combine relevant databases and literature information to provide explanations and reports of biological significance.
The NGS experimental process is a complex and detailed process, covering multiple links from DNA extraction, library construction, targeted capture (optional), computer sequencing to data analysis, etc. Each step is critical and requires strict technical operation and quality control to ensure the accuracy and reliability of sequencing results.
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