Next-generation Sequencing Systems
Next generation sequencing (NGS) is a technology for determining DNA or RNA sequences on a large scale, also known as high-throughput, massively parallel, and deep sequencing. NGS can sequence millions of nucleic acid fragments at once, providing detailed information on genome structure, genetic variation, gene activity, and changes in gene behavior. First-generation sequencing, also known as Sanger sequencing, is a DNA sequencing technique based on selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during DNA replication in vitro. The main limitation of first-generation sequencing is the low throughput capacity. Compared to first-generation sequencing, NGS technology is designed to enable faster and more accurate sequencing, reduce costs, and improve data analysis. NGS enables rapid sequencing of millions of nucleic acid fragments simultaneously, providing a comprehensive understanding of genome structure, genetic variation, gene expression profiles, and epigenetic modifications.
Short-read NGS, or second-generation sequencing methods, have revolutionized DNA sequencing by enabling the simultaneous sequencing of thousands to millions of DNA fragments. The common feature of short-read techniques is the massive parallel sequencing of short (250-800 bp) clonally amplified DNA molecules. The basic principle of short-read sequencing involves synthetic sequencing based on enrichment through hybridization, amplification, or fragment. In contrast to second-generation sequencing methods, third-generation sequencing methods aim to sequence long DNA and RNA molecules. These sequencing methods provide long-read sequencing capabilities, capable of sequencing much larger DNA segments compared to earlier methods. Commonly, long-read sequencing can produce more than 10 kb of reads. Long-read sequencing works on sequence detection by synthesis or voltage change/impedance, which generates current as a single base is passed through the biological membrane pore. Theoretically, in nanopore sequencing, more than 100 kb of DNA or RNA can pass through the nanopore, and there are many nanopores in each channel, so tens to hundreds of Gb of sequence can be achieved simultaneously at relatively low cost. Since the library preparation is PCR-free, using long-read sequencing methods can easily detect base modifications such as DNA methylation. However, the error rate of long-read sequencing is higher than that of short-read sequencing. Short-read NGS can be used to count the abundance of specific sequences, profile transcript expression, and identify the variants, while long-read NGS can be used to identify complex structural variants such as large insertions, deletions, reversals, repeats, deletions, etc.
The NGS system is an important instrument in modern laboratories for efficient and rapid sequencing of large-scale DNA or RNA molecules. The NGS workflow includes library preparation, sequencing, and data analysis. The choice of the sequencer depends on many factors such as the laboratory throughput, sample type, and flexibility required to run pilot tests. Amerigo Scientific offers high-performance sequence devices, as well as compatible reagents and consumables.
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