Introduction
In the past ten years, the discipline of genomics has improved significantly thanks to the groundbreaking technique known as next-generation sequencing (NGS). The automated, high-throughput NGS method makes it possible to sequence DNA or RNA quickly and affordably. Using this technology, it is now able to sequence complete genomes, find mutations, and evaluate gene expression levels quickly and inexpensively compared to earlier techniques. The fundamentals of NGS, important phases in the procedure, various NGS technology types, and some of the fascinating NGS applications will all be covered in this blog article.
The Basics of Next-Generation Sequencing
The steps involved in NGS include preparing a DNA or RNA sample, fragmenting the sample, sequencing the fragments, and assembling the sequence data. A sample, such as blood, saliva, or tissue, is often used to begin the procedure by extracting the DNA or RNA from it. The extracted sample is then fragmented into small fragments for sequencing using enzymes or sonication. After being ligated using adapters, the fragments may now be amplified and sequenced.
A sequencing platform, such as one from Illumina, Pacific Biosciences, or Oxford Nanopore, is used to load the material once it has been prepared. The fundamental idea behind all of these platforms, which employ various sequencing chemistries, is the same: each fragment is amplified into a cluster of identical sequences, and the sequence of each fragment is determined by a series of chemical reactions that produce a colour signal that is detected by a camera.
After that, the sequencing data is examined to find genetic variations, levels of gene expression, and other characteristics of the genome or transcriptome. In most analyses, the sequence reads are aligned to a reference genome, variations or differences between the sample and the reference are found, and the biological relevance of the differences is determined.
The Different Types of Next-Generation Sequencing Technologies
NGS technologies come in a variety of forms, each with distinct advantages and disadvantages.
- Illumina sequencing,
- Pacific Biosciences (PacBio) sequencing, and
- Oxford Nanopore sequencing
are the most frequently utilised technologies.
- The most used NGS technology is Illumina sequencing, which produces short reads using a sequencing-by-synthesis process (typically 100-300 base pairs). Illumina sequencing is efficient, precise, and reasonably priced, but its capacity to sequence lengthy segments or identify specific forms of genetic variation is constrained.
- In comparison to Illumina sequencing, PacBio sequencing use single-molecule real-time (SMRT) sequencing technology, which produces longer reads (up to 100,000 base pairs) and has a better accuracy rate. Although PacBio sequencing is somewhat slow and costly, it is ideally suited for de novo genome assembly, structural variation identification, and study of epigenetic changes.
- Long fragments (up to 2 million base pairs) may be quickly and accurately sequenced using Oxford Nanopore sequencing, which is based on nanopore technology. By making it possible to directly sequence complicated genomic areas including repeat sequences, structural variants, and epigenetic changes, this technique has the potential to transform genomics research. Oxford Nanopore sequencing is still less accurate than Illumina and PacBio sequencing, and the cost of the technology is still high.
Next-generation sequencing applications
NGS has had a substantial influence on a number of disciplines, including environmental studies, agriculture, and medical research. Listed here are a few NGS applications:
- Medical research: Genetic mutations that cause illnesses including cancer, cystic fibrosis, and Alzheimer's disease have been found using NGS. In order to better understand the genetic causes of health and illness, it was also utilised to sequence the human genome.
- Forensic science: By sequencing criminals' DNA from crime scenes, NGS has been utilised in forensic science to identify offenders.
- Agriculture: Crop yields have been increased thanks to the use of NGS, which has also been used to find genetic markers for breeding schemes.
- Environmental research: To detect microbial communities and explore biodiversity, NGS has been utilised in environmental studies.
Methods for Next-Generation Sequencing
There are various NGS methods available, and each has benefits and drawbacks. Here are a few of the most popular NGS methods:
- The most used NGS approach is Illumina sequencing. High-throughput sequencing and accurate base calling are made possible by the use of reversible terminators.
- Ion Torrent Sequencing: Using semiconductor sequencing, this method tracks the release of hydrogen ions as each nucleotide joins the DNA chain.
- PacBio sequencing: This method employs single-molecule real-time (SMRT) sequencing, allowing for long-read sequencing, which makes it perfect for de novo genome assembly.
Developments in Next-Generation Sequencing in the Future
Even though NGS has changed the area of genomics, more can be done. The following are some future developments that we can anticipate:
- Long-read sequencing: Although while NGS has significantly increased the efficiency of sequencing, it still has trouble completing lengthy DNA segments. PacBio and Oxford Nanopore's long-read sequencing technologies have showed promise in this field and might see more use in the future.
- Single-cell sequencing: Because current NGS technologies need a lot of starting material, sequencing individual cells is difficult. In order to sequence the DNA of individual cells and better comprehend cellular heterogeneity, single-cell sequencing methods are being developed.
- Integration with other technologies: CRISPR-Cas9 and artificial intelligence are two examples of other technologies that are now employed in combination with NGS. In the future, we may anticipate further integrations that will enable us to get even more information from genetic data.
Conclusion
The discipline of genomics has been completely transformed by NGS technologies, which allow scientists to investigate an organism's genetic makeup more quickly and precisely than ever before. NGS offers a wide range of uses in both academic and medical contexts, including cancer research, the study of human genetics, and the identification of genetic illnesses. Even if there are still issues that need to be resolved, NGS is still making progress because to the continual creation of new tools and techniques.
