Next-Generation Sequencing (NGS), or Second-Generation Sequencing, is a high-throughput technology used in molecular biology to rapidly sequence DNA or RNA. NGS techniques have revolutionized genomics research, enabling scientists to sequence multiple genes quickly and at a relatively low cost compared to traditional mutation detection methods.
The key idea behind NGS is its massive throughput by generating millions of short DNA fragments in parallel. The most popular technology is sequencing by synthesis, which was leveraged by Illumina to produce approximately 90% of the current world's sequence data. This approach relies on four fluorescently-labeled nucleotides to sequence millions of clusters on the flow cell surface in a parallel fashion.
The Evolution of NGS
First-Generation Sequencing
Fredrick Sanger's chain-termination sequencing, developed in the 1970s, revolutionized molecular biology. It enabled the sequencing of short DNA fragments and paved the way for automated sequencing methods like the ABI 370, which significantly improved throughput. However, first-generation methods were time-consuming and expensive for large-scale projects, limiting their use in genome-wide studies.
Second-Generation Sequencing
The leap to second-generation sequencing (also known as NGS) introduced parallel sequencing, where millions of DNA fragments could be sequenced at the same time. Platforms such as Illumina, Roche’s 454, and Ion Torrent made it possible to perform whole-genome sequencing, whole-exome sequencing, and targeted sequencing. These methods not only improved speed but also lowered costs, making high-throughput sequencing more accessible to researchers.
Third-Generation Sequencing
Third-generation sequencing technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore, further improved NGS by enabling long-read sequencing. This allows researchers to sequence larger DNA fragments, up to tens of kilobases, overcoming the limitations of earlier short-read technologies. Long-read sequencing is particularly useful for genome assembly and identifying complex structural variants such as large insertions, deletions, and duplications.
NGS-Based Omics Technologies
Genomics
Genomics studies using NGS provide comprehensive analyses of DNA, allowing researchers to sequence entire genomes (whole-genome sequencing) or focus on specific coding regions (whole-exome sequencing). These approaches are critical in identifying genetic variations, such as single-nucleotide polymorphisms (SNPs), copy number variations (CNVs), and structural variants. NGS-based genomics has advanced the study of rare diseases, cancer, and population genetics.
Transcriptomics
In transcriptomics, NGS is used to sequence RNA molecules, offering a snapshot of gene expression in specific cells or tissues. The most common NGS application in transcriptomics is RNA sequencing (RNA-seq), which quantifies mRNA levels, identifies alternative splicing events, and detects novel transcripts. This has provided invaluable insights into gene expression dynamics, regulatory networks, and disease mechanisms. Additionally, NGS enables the study of non-coding RNAs, such as microRNAs and long non-coding RNAs (lncRNAs), which play critical roles in gene regulation.
Epigenomics
NGS is also a key technology in epigenomics, which studies heritable changes in gene expression that do not involve alterations in the DNA sequence. Epigenomic analyses focus on modifications like DNA methylation and histone modifications, which regulate gene activity and cellular differentiation. NGS-based techniques, such as bisulfite sequencing, allow researchers to map methylation patterns across the genome, providing insights into gene regulation during development and disease processes such as cancer.
Metagenomics
Metagenomics involves the study of microbial communities in various environments, from the human gut to the ocean. Using NGS, researchers can sequence the genetic material of entire microbial populations without needing to culture individual species. This has enabled the identification of microbial diversity and the study of how microbes interact with each other and their hosts. Metagenomics is especially important in understanding the role of the microbiome in health and disease, including its impact on conditions like diabetes, obesity, and autoimmune disorders.
As technology continues to advance, the future of NGS looks incredibly promising. Innovations in bioinformatics, robotics, and sequencing chemistry are expected to make NGS even faster, more accurate, and cost-effective. The emergence of real-time sequencing, single-cell genomics, and long-read sequencing will expand NGS applications in clinical diagnostics and basic research. Additionally, as bioinformatic tools improve, researchers will be able to integrate multi-omics data more effectively, providing a systems-level understanding of complex diseases.
In the coming years, NGS is likely to become even more portable, with devices that can be used in field-based settings for medical, agricultural, and environmental applications. With these advancements, NGS will continue to drive discoveries in genomics and revolutionize personalized medicine, agriculture, and biotechnology.
References:
Satam, H., Joshi, K., Mangrolia, U., Waghoo, S., Zaidi, G., Rawool, S., Thakare, R. P., Banday, S., Mishra, A. K., Das, G., & Malonia, S. K. (2023). Next-Generation Sequencing Technology: Current Trends and Advancements. Biology, 12(7). https://doi.org/10.3390/biology12070997
