Introduction
Next Generation Sequencing (NGS) Technologies are powerful tools used in genomics research providing detailed information about the structure of genomes, genetic variations, gene activity, and changes in gene behavior.(Satam et al., 2023). It provides high-throughput, accurate, and cost-effective solutions for direct sequencing. It accelerated the pace of new genome sequencing. Applications in genomics, transcriptomics, epigenomics, and metagenomics have provided tremendous benefits in clinical settings. This article provides an overview of Next-generation sequencing (NGS) technologies and their applications, such as in genomics, cancer, epigenomics, metagenomics, transcriptomics, etc.
The need for NGS technologies
Next-generation sequencing (NGS) technologies have become indispensable in genomics research and various fields due to several key advantages over traditional sequencing methods like Sanger sequencing. Here are some of the critical needs that NGS technologies address:
- Direct sequencing method
- high-throughput, accurate, and reproducible
- cost-effective
- Advances in Epigenetics
- Applications in Transcriptomics and Proteomics
Next Generation Sequencing Technologies
- Roche 454 Sequencing (currently not available)
- Illumina sequencing by synthesis (SBS) technology
- Pacific Biosciences Single-Molecule Real-Time (SMRT) sequencing
- Ion Torrent sequencing
- Oxford Nanopore sequencing
| Sr No. | Platform | Use | Sequencing Technology | Amplification Type | Read Length (bp) |
|---|---|---|---|---|---|
| 1 | 454 pyrosequencing | Short-read sequencing | Seq by synthesis | Emulsion PCR | 400–1000 |
| 2 | Ion Torrent | Short-read sequencing | Seq by synthesis | Emulsion PCR | 200–400 |
| 3 | Illumina | Short-read sequencing | Seq by synthesis | Bridge PCR | 36–300 |
| 4 | SOLiD | Short-read sequencing | Seq by ligation | Emulsion PCR | 75 |
| 5 | DNA nanoball sequencing | Short-read sequencing | Seq by ligation | Amplification by Nanoball PCR | 50–150 |
| 6 | Helicos single-molecule sequencing | Short-read sequencing | Seq by synthesis | Without Amplification | 35 |
| 7 | PacBio Onso system | Short-read sequencing | Seq by binding | Optional PCR | 100–200 |
| 8 | PacBio Single-molecule real-time sequencing (SMRT) technology | Long-read sequencing | Seq by synthesis | Without PCR | 10,000–25,000 |
| 9 | Nanopore DNA sequencing | Long-read sequencing | Sequence detection through electrical impedance | Without PCR | 10,000–30,000 |
| Sr No. | Platform | Principle | Limitations |
|---|---|---|---|
| 1 | 454 pyrosequencing | Detection of pyrophosphate released during nucleotide incorporation. | May contain deletion and insertion sequencing errors due to inefficient determination of homopolymer length. |
| 2 | Ion Torrent | Ion semiconductor sequencing principle detecting H+ ion generated during nucleotide incorporation. | When homopolymer sequences are sequenced, it may lead to loss in signal strength. |
| 3 | Illumina | Solid-phase sequencing on immobilized surface leveraging clonal array formation using proprietary reversible terminator technology for rapid and accurate large-scale sequencing using single labeled dNTPs, which are added to the nucleic acid chain. | In case of sample overloading, the sequencing may result in overcrowding or overlapping signals, spiking the error rate up to 1%. |
| 4 | SOLiD | Enzymatic method of sequencing using DNA ligase. 8-Mer probes with a hydroxyl group at 3′ end and a fluorescent tag (unique to each base A, T, G, C) at 5′ end are used in the ligation reaction. | Displays substitution errors and may under-represent GC-rich regions. Short reads also limit broader applications. |
| 5 | DNA nanoball sequencing | Splint oligo hybridization with post-PCR amplicon from libraries forms circles. Circular ssDNA is used as the DNA template to generate long strings that self-assemble into DNA nanoballs. These are bound to amino silane-coated flow cells. | Multiple PCR cycles and exhaustive workflow limit efficiency. |
| 6 | Helicos single-molecule sequencing | Poly-A-tailed short 100–200 bp fragmented genomic DNA sequenced on poly-T oligo-coated flow cells using fluorescently labeled 4 dNTPs. The signal released upon nucleotide addition is captured. | Highly sensitive instrumentation is required. As sequence length increases, fewer strands can be utilized. |
| 7 | PacBio Onso system | Sequencing by binding (SBB) chemistry uses native nucleotides and scarless incorporation under optimized conditions for binding and extension. | Higher cost compared to other platforms. |
| 8 | PacBio Single-molecule real-time sequencing (SMRT) technology | SMRT sequencing employs SMRT Cell housing zero-mode waveguides (ZMWs). DNA molecules immobilized in ZMWs emit light during polymerase activity, allowing real-time nucleotide incorporation. | Higher cost compared to other platforms. |
| 9 | Nanopore DNA sequencing | DNA or RNA molecules pass through biological nanopores (8 nm wide). Electrophoretic mobility generates a current signal as linear nucleic acid strands pass through. | Error rates can spike to 15%, especially with low-complexity sequences. Lower accuracy compared to short-read sequencers. |
Benefits of NGS vs. Sanger Sequencing
- Higher sensitivity to detect low-frequency variants
- Faster turnaround time for high sample volumes
- Comprehensive genomic coverage
- The lower limit of detection
- Higher capacity with sample multiplexing
- Ability to sequence hundreds to thousands of genes or gene regions simultaneously[1]
Applications of Next-Generation Sequencing (NGS) Technologies
Application in Genomics
Applications in Transcriptomics
- Measurement of the expression level of all genes
- Sequencing all mRNA molecules (transcriptome) of a Cell
- Discovery of novel transcripts
- Alternative splicing
- Investigating disease states, for example, in cancer
The use of transcriptomics has transformed our understanding of human diseases
The Cancer Genome Atlas (TCGA) Project https://portal.gdc.cancer.gov/
Applications in Epigenomics
Epigenetics is the study of the chemical modification of specific genes or gene-associated proteins of an organism. Epigenetic modifications can define how gene information is expressed and used by cells(Fridovich-Keil et al, Encyclopedia Britannica). The most common epigenetic modifications studied are DNA methylation, histone modification, and RNA methylation.
- Identifying changes in epigenetic modifications in diseases
- Titanji et al., Epigenome-wide epidemiologic studies of human immunodeficiency virus infection, treatment, and disease progression. Clin Epigenetics. 2022; 14:8.
NGS has been utilized for investigating epigenomics:
- DNA Methylation Profiling
- Chromatin Accessibility Mapping
- Histone Modification Analysis
- Chromatin Conformation Analysis
- NGS data from epigenomics can be integrated with transcriptomics data to understand the relationship between epigenetic modifications and gene expression.
Applications in Metagenomics
Metagenomics has transformed the study of microbial communities, enabling a deeper understanding of complex systems such as the soil microbiome and gut microbiome. These advances have significant clinical implications, including the potential for disease detection, informed decisions regarding treatment choice, and the development of targeted therapies. Additionally, metagenomics plays a crucial role in environmental sciences, particularly in bioremediation, where microbial processes are harnessed to detoxify or restore contaminated environments.
Machine Learning Approaches in Genomics
The ever-increasing availability of large datasets in genomics, transcriptomics, and epigenomics has catalyzed the development of machine learning models. These models are designed to leverage vast amounts of biological data for classification and prediction tasks. For instance, machine learning approaches are employed to predict cancer sub-type classifications from transcriptomic data, offering more precise diagnostic tools.
Benefits of NGS Technologies
Next-generation sequencing (NGS) technologies offer several advantages that have revolutionized genomics research. These include the ability to sequence thousands of genes or genomic regions simultaneously and to directly sequence unknown genomic fragments or entire genomes. NGS also facilitates the high-throughput sequencing of large sample sizes in a relatively short period, enhancing the ability to detect low-frequency variants. Furthermore, NGS is highly cost-effective, especially for processing large datasets.
References
- [1] NGS vs. Sanger Sequencing (illumina.com)
- Gupta N, Verma VK. Next-Generation Sequencing and Its Application: Empowering in Public Health Beyond Reality. Microbial Technology for the Welfare of Society. 2019 Sep 13;17:313–41. doi: 10.1007/978-981-13-8844-6_15. PMCID: PMC7122948.
- 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), 997. https://doi.org/10.3390/biology12070997
- Fridovich-Keil, Judith L. and Rogers, Kara. “epigenetics”. Encyclopedia Britannica, 27 Jul. 2024, Accessed 8 September 2024.
