Second-generation sequencing

Next-generation sequencing (NGS), also known as high-throughput sequencing, is a DNA sequencing technology that has evolved from PCR and gene chips. Compared to the first-generation sequencing method, which relied on chain-termination sequencing, NGS pioneered the use of reversible terminator nucleotides, enabling simultaneous synthesis and sequencing. During DNA replication, NGS determines the DNA sequence by detecting special markers—typically fluorescent labels—attached to newly added nucleotides. Current major NGS platforms include Ion Torrent, Illumina, and BGI’s MGI platform. In NGS, individual DNA molecules must be amplified into clusters composed of identical DNA sequences and then replicated synchronously to enhance the fluorescence signal strength and facilitate DNA sequence readout. However, as read lengths increase, the coordination of cluster replication becomes less efficient, leading to a decline in base-call accuracy. This limitation severely restricts the maximum read length of NGS to no more than 500 base pairs. Consequently, NGS is characterized by high throughput but relatively short read lengths. Currently, NGS primarily includes two main applications: metagenomic next-generation sequencing (mNGS) and targeted next-generation sequencing (tNGS). mNGS leverages NGS platforms to rapidly sequence nucleic acid fragments from a sample and then compares these sequences against reference genomes of various species, thereby identifying the types and relative abundances of microorganisms present in the sample. mNGS offers several advantages: comprehensive detection, high accuracy, high sensitivity, and rapid turnaround time. When identifying pathogenic microorganisms in a sample, mNGS can complement or even replace traditional biochemical, immunological, and culture-based methods, providing faster and more accurate results. More importantly, conventional methods often prove ineffective when dealing with rare, uncommon, or emerging infections. By detecting all microbial species present in a sample, mNGS can pinpoint potential pathogens and guide appropriate antibiotic treatment. At the same time, mNGS does have certain limitations. First, since human-derived nucleic acids often constitute a large proportion of clinical samples, the sensitivity of pathogen detection is reduced, increasing the risk of missed detection of drug resistance and virulence genes. Second, because mNGS involves random fragment sequencing, the sequencing process may not always cover polymorphic regions, potentially leading to imprecise identification of closely related pathogens.

tNGS is a high-throughput sequencing technology that focuses exclusively on specific gene sequences. It employs a large number of primer probes designed to target particular gene sequences, enabling highly multiplex PCR amplification and probe capture of nucleic acids extracted from the sample under analysis. This process yields a large quantity of target nucleic acid fragments, which are then subjected to high-throughput sequencing. Subsequently, bioinformatics analysis is performed on the resulting sequences, allowing for highly sensitive and high-resolution identification of nucleic acids in the sample being tested. Target enrichment is a critical step in tNGS technology; by enriching regions of interest (ROIs) in the genome, it ensures that these ROIs achieve sufficient sequencing depth and coverage, thereby enabling successful identification of target pathogens. Currently, both domestic and international approaches mainly fall into two categories: tNGS based on ultra-multiplex PCR amplification and tNGS based on hybridization capture. Compared to mNGS, tNGS offers the following technical advantages: 1. Enrichment increases the coverage of target microbial detection, enhancing the reliability of results; 2. NGS enables stable detection of drug resistance or virulence genes; 3. Since tNGS targets specific pathogen ranges with clearly defined scopes, it can significantly simplify the interpretation process. However, tNGS also has certain limitations: the detection panels used in tNGS are designed based on known pathogens, so tNGS is not suitable for detecting novel pathogens.

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