Basic steps of NGS detection

NGS (Next Generation Sequencing Technology) is a revolutionary genomics technology that can efficiently and accurately sequence the genome on a large scale, and is widely used in many fields such as genomics, medical diagnosis, and agricultural research. The basic steps of NGS detection cover the entire process from sample preparation to data analysis.

NGS Detection

1. Sample collection and pretreatment1. Sample collection: According to the purpose of detection, collect appropriate biological samples, such as blood, tissue, saliva, etc. 2. Sample pretreatment: Perform necessary treatment of collected samples, such as extracting DNA or RNA, removing impurities, and ensuring that the sample quality meets sequencing requirements.

2. Library construction1. DNA/RNA fragmentation: Cut the extracted DNA or RNA into short fragments suitable for sequencing, usually in length of tens to hundreds of base pairs. 2. Linker connection: Add sequencing adapters to both ends of the fragment, which contain the primer sequences required during the sequencing process and the tags identified by the sequencing machine. 3. Amplification: Increase the number of DNA fragments in the library by PCR or other amplification methods to obtain sufficient signals during sequencing.

3. Sequencing preparation1. Library quality detection: Use biological analysis instruments to detect the quality of the library, including fragment size distribution, concentration, etc. 2. Sequencing platform selection: Based on the sequencing needs and cost considerations, select suitable sequencing platforms, such as Illumina, Ion Torrent, PacBio, etc.

IV. Sequencing reaction1. Preparation of sequencing chip or sequencing flow cell: Load the library into the sequencing chip or sequencing flow cell to prepare for sequencing reactions. 2. Sequencing reaction: In a sequencing machine, the sequence information of DNA fragments is read base-by-base by base-by-base sequencing (SBS) or other sequencing methods. During the sequencing process, the sequencing instrument will record the signal strength of each base for subsequent data analysis.

5. Data Analysis1. Raw data processing: Quality filtering and denoising the original data generated by the sequencing instrument (such as FASTQ files) to improve data quality. 2. Sequence alignment and assembly: Aligate the filtered sequences with the reference genome, or assemble without reference genome to obtain the genome sequence information of the sample. 3. Variation detection: Through the comparison results, genetic variations such as single nucleotide polymorphisms (SNP), insertion/deletion (INDEL), structural variations in the sample are detected. 4. Bioinformatics analysis: Based on the detected mutation information, gene function annotation, pathway analysis, disease association analysis, etc. are carried out to provide scientific basis for subsequent medical diagnosis, drug development, etc.

6. Report and Interpretation1. Generate a report: organize the sequencing results and analysis results into reports, including sample information, sequencing quality, variation detection results, etc. 2. Results interpretation: According to the content of the report, combined with clinical or research background, the sequencing results are interpreted and corresponding medical suggestions or research conclusions are put forward.

NGS detection technology covers several crucial basic steps, including sample collection and preprocessing, library construction, sequencing preparation, sequencing reactions, data analysis, and final report interpretation. Each link has a profound impact on the accuracy and reliability of sequencing results. With the continuous advancement of technology, NGS detection has shown broad application prospects in many fields such as genomics, personalized medicine, and disease diagnosis.

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