Next-generation sequencing (NGS), sometimes referred to as “deep sequencing” or “massively parallel sequencing,” is a DNA sequencing technique that has paved the way for major progression in clinical and translational research. Clinicians and researchers use the technique to study the underlying DNA mechanisms associated with neonatal and infectious diseases, rare genetic disorders, cancers, and more. Studies into these diseases and disorders have laid the groundwork for the development of better-targeted, personalized therapies.
Although next-generation sequencing offers plenty of benefits, both in the clinic and in research, it doesn’t come without its disadvantages. The life sciences journal BioTechniques is a good place to read up on these pros and cons. The journal is widely recognized for covering the efficacy and reproducibility of techniques and methods in science and medicine, rather than focusing wholly on treatments.
Here, we’ll work through the stages of NGS and discuss its pros and cons and its role in a variety of applications.
The Stages of Next-Generation Sequencing
The NGS workflow involves four main stages. The first is sample preparation, during which a researcher extracts genomic DNA from a sample. The sample is usually saliva, blood, or tissue. The researcher fragments the DNA into shorter sequences and follows up with ligation of adapters, amplification, and enrichment.
The next stage is library preparation. During this stage, the researcher randomly fragments the DNA or cDNA. They usually achieve this through sonication or by applying an enzymatic treatment. The platform they use dictates the optimum fragment length.
The third stage is the sequencing stage. The researcher chooses a sequencing method based on their platform. Some example methods include pyrosequencing, sequencing by synthesis or ligation, and reversible terminator sequencing. Sequencing by synthesis is one of the most commonly selected methods as this approach enables researchers to sequence lots of DNA at once and at high sensitivity. With this approach, researchers can detect a large range of genetic alterations, such as structural variants, small insertions and deletions, and single-nucleotide polymorphisms (SNPs).
The final stage of the workflow is data analysis. The researcher uses data analysis applications or bioinformatic tools to pinpoint pathogenic variants, align to the reference sequence, and perform quality control checks.
Pros of Next-Generation Sequencing
Before scientists developed NGS, they performed Sanger (first-generation) sequencing, which was prevalent for 30 years and formed the basis of scientists’ understanding of the human genome. Now, NGS has superseded Sanger sequencing because it offers a more efficient workflow, improved sensitivity, and coverage. It’s also much more cost-effective: The cost of sequencing the human genome has fallen over the past decade from $20-$25 million to under $1,000 by 2016.
The time required to carry out NGS and receive results has also dropped over recent years. Today, NGS platforms can sequence millions of DNA fragments simultaneously, meaning that researchers can sequence virtually anything within a day, from specific target regions to the entire human genome. It takes approximately 10 days to receive a whole-genome sequencing report from the day the lab receives the tumor specimen.
On top of this, NGS makes it possible for researchers to identify abnormalities across the whole genome. This means they can sequence abnormalities across insertions, deletions, substitutions, duplications, chromosome inversions/translocations, and copy number changes (gene and exon). NGS can also identify abnormalities across the entire genome using less DNA than the DNA required for traditional sequencing methods.
Cons of Next-Generation Sequencing
Despite all the benefits of NGS, the technique does have some cons. First, although NGS provides information on many molecular aberrations, the clinical significance of many identified abnormalities is still unknown.
Second, NGS requires large data storage capabilities, sophisticated bioinformatics systems, and fast data processing infrastructures, each of which can be costly. Although some institutions have the funding to meet these requirements, others can’t fund the staff or computational resources to interpret and analyze such high data loads.
Third, while researchers can use NGS to sequence a whole DNA sequence, they can only use data from approximately 3% of the genome in clinical practice. So, NGS has much more potential in the research space than it does in the clinical space.
Next-Generation Sequencing Applications
Despite NGS’ limitations, the technique has become integral to much medical and scientific research. Scientists have employed NGS in several research applications, such as whole-genome sequencing to identify an organism’s complete DNA sequence, whole-exome sequencing to analyze a genome’s coding regions, targeted sequencing to examine specific genomic regions, epigenomics to assess epigenetic modifications, and PCR for next-generation polymerases in the NGS workflow. Researchers have also used NGS in RNA sequencing to perform transcriptome profiling of coding and non-coding regions, identify genes in specific cell types, and determine genetic alterations such as gene fusions and single nucleotide variants (SNVs).
The latest developments in NGS have even seen the technique advance therapies that enhance COVID-19 testing and combat obesity. As scientists continue to shape NGS technologies, sequencing techniques should become even more cost-effective and accessible.
Publishing Advances in Life Science Technologies
BioTechniques has been reviewing laboratory methods and techniques since 1983. Since then, the journal has grown a global audience of scientists and research professionals who specialize in fields spanning from the life sciences, chemistry, and physics to computer science, plant science, and agricultural science. Not only do these users benefit from the print journal, but they also find a wealth of resources on BioTechniques’ multimedia website. These resources include articles, eBooks, videos, interviews, webinars, and podcasts, which delve into laboratory methods like next-generation sequencing, western blotting, polymerase chain reaction, chromatography, and CRISPR gene editing.
BioTechniques is one of the 34 peer-reviewed, open-access journals that Future Science Group publishes. The progressive medical and scientific publisher is also home to titles like Regenerative Medicine, Nanomedicine, and Future Oncology. Future Science Group receives over 5 million article downloads every year and has published over 50,000 articles so far.
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