Next generation sequencing

Next-generation sequencing (NGS), also known as high-throughput sequencing (High-throughput sequencing), is a DNA sequencing technology based on PCR and gene chips. In contrast to the first-generation sequencing method which relied on synthetic termination sequencing, second-generation sequencing has pioneered the introduction of reversible termination ends, which can realize sequencing by synthesis. Second-generation sequencing determines the sequence of DNA by capturing special markers (usually fluorescent molecular markers) carried by newly added bases during DNA replication. The existing second-generation sequencing technology platforms mainly include Ion torrent, Illumina and Huada MGI platform. In the second generation sequencing, a single DNA molecule must be amplified into a gene cluster composed of the same DNA, and then synchronized to enhance the fluorescence signal intensity to read the DNA sequence. However, with the growth of reading length, the synergy of gene cluster replication decreases, resulting in the decline of base sequencing quality, which strictly limits the reading length of the second generation sequencing (no more than 500bp). Therefore, the second generation sequencing has the characteristics of high throughput and long reading. At present, next generation sequencing (NGS) mainly includes metagenome second generation sequencing technology (metagenomics next-generation sequencing, mNGS) and pathogen targeted sequencing technology (Targeted next-generation sequencing,tNGS). mNGS is a technology for rapidly sequencing the nucleic acid sequence in the sample by means of the second generation sequencing platform, and further comparing with the genome sequence of each species, so as to know the type and proportion of microorganisms in the sample. mNGS has the advantages of comprehensive detection, high accuracy, high sensitivity and short time. When identifying pathogenic microorganisms in samples, mNGS can supplement or replace traditional biochemical, immune and culture methods to obtain faster and more accurate results. More importantly, traditional methods are often helpless in dealing with rare, uncommon infections and new infections. mNGS detects all microbial species in samples to identify potential pathogenic microorganisms and guide medication. At the same time, mNGS has some limitations. First of all, due to the high proportion of human nucleic acids in clinical samples, the sensitivity of pathogenic microorganism detection is reduced, and the risk of missing detection of drug resistance and virulence genes is also caused. Secondly, because mNGS sequencing is random fragment sequencing, sequencing may not cover the typing segment, which may lead to the inability to accurately type near-source pathogens.

tNGS is a high-throughput sequencing technology only for specific gene sequences. Through a large number of primers and probes for specific gene sequences, the nucleic acids extracted from the samples to be tested are amplified by ultra-multiplex PCR/probe capture, and a large number of target nucleic acid fragments are obtained. Then, high-throughput sequencing is carried out on these target nucleic acid fragments, and then the obtained sequences are analyzed, thus, highly sensitive and high-resolution identification of the nucleic acid in the sample to be detected is achieved. Target enrichment is the key link of tNGS technology, through the target genomic region (regions of interest ,ROI) enrichment, to ensure that the ROI region has sufficient sequencing depth and coverage, and then successfully identify the target pathogen. At present, there are two main strategies at home and abroad: tNGS based on super-multiplex PCR amplification and tNGS based on hybrid capture. Compared with mNGS,tNGS has the following technical advantages: 1. Enrichment increases the detection coverage of target microorganisms and improves the detection reliability; 2. NGS can stably detect drug resistance or virulence genes; 3. tNGS detection can significantly simplify the interpretation process due to the clear setting range of target pathogens. However, tNGS also has some limitations. The detection panel of tNGS is designed according to the known pathogens, so tNGS is not suitable for the detection of new pathogens.

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