DNA sequencing - next generation sequencing

First introduced for commercial use in 2005, next-generation sequencing (NGS) is a technology that scientists utilize to determine the sequence of DNA or RNA. Identifying such sequences can be essential to the study of genetic variation associated with certain diseases or other biological phenomena. Next-generation sequencing (NGS) is sometimes known as “deep sequencing” or “massively parallel sequencing.” The technology gained this name because it allows scientists to sequence multiple strands of DNA at once, rather than one by one through the traditional Sanger sequencing method.

As NGS enables developments in both clinical and translational research, and in the foundations of better-targeted, personalized treatments, many research professionals keep up with these sequencing developments by following BioTechniques, the award-nominated life sciences journal. Here, we will touch on the advantages and disadvantages of NGS, both of which BioTechniques explores in detail in its journal and on its multimedia website.

The Four Steps of Next-Generation Sequencing 

Scientists completing NGS must work through four steps: sample preparation, library preparation, sequencing, and data analysis.

  1. During the sample preparation, the scientist extracts genomic DNA from a sample, which may be tissue, blood, or saliva. The scientist fragments the DNA into shorter sequences and finishes this step with the ligation of adapters, amplification, and enrichment.
  2. The library preparation step sees the scientist randomly fragment the DNA or cDNA, typically by applying an enzymatic treatment or through sonication. The platform they use informs the optimal fragment length.
  3. The scientist moves on to the sequencing stage, in which they select a sequencing method based on their platform. They may choose a method like sequencing by synthesis or ligation, pyrosequencing, or reversible terminator sequencing. Sequencing by synthesis is one of the most frequently employed methods. This type of sequencing allows scientists to sequence high volumes of data in parallel and at high sensitivity. This way, they can identify a large range of genetic alterations, like single-nucleotide polymorphisms (SNPs), structural variants (SVs), and small deletions and insertions.
  4. Finally, the scientist completes the data analysis phase of the workflow. They implement bioinformation tools or data analysis applications to pinpoint pathogenic variants, conduct quality control checks, and align to the reference sequence.

Advantages of Next-Generation Sequencing

Before the advent of NGS, scientists usually employed Sanger, or first-generation, sequencing. This type of sequencing was dominant for three decades and allowed scientists to develop their understanding of the human genome. Today, NGS has superseded this type of sequencing and offers a more modern approach with improved sensitivity and coverage, a more effective workflow, and a lower cost. In fact, with NGS, the cost of sequencing the human genome fell over 10 years from $20-$25 million to under $1,000 by 2016.

As NGS has developed over recent years, the time required to sequence DNA has reduced. Scientists can now sequence millions of DNA fragments at the same time. They can also sequence virtually anything, from specific target regions to the entire human genome, within 24 hours. After a laboratory receives a tumor specimen,it takes approximately 10 days to receive a whole-genome-sequencing report.

Furthermore, with NGS, scientists can recognize irregularities across a whole genome. As a result, they can sequence abnormalities across deletions, insertions, duplications, substitutions, chromosome translocations/inversions, and copy number changes, which may be gene or exon. Scientists can also recognize these abnormalities using less DNA than the DNA needed to complete traditional sequencing methods.

Disadvantages of Next-Generation Sequencing

Although NGS offers many advantages and has accelerated research and clinical developments in the gene editing space, there are some disadvantages of this type of sequencing. First, NGS relies on advanced bioinformatics systems, fast data processing infrastructures, and large data storage capabilities, all of which can be costly. Some institutions have funding for these, but others may not be able to fund the resources or staff needed to interpret and analyze high data loads. 

Meanwhile, although scientists can employ NGS to sequence an entire DNA sequence, they can only analyze data from approximately 3% of the genome in clinical practice. Therefore, NGS currently offers much more potential in research than it does in clinical practice. Finally, while NGS reveals information on several molecular aberrations, scientists still don’t understand the clinical significance of many of the abnomalities that have been identified.

Applications of Next-Generation Sequencing

While it’s important to note that NGS does come with its disadvantages, we can celebrate the fact that the technology has proven crucial to a high volume of scientific and medical research. Scientists have leveraged NGS in a variety of research applications, in which the technology has enabled them to:

  • Analyze a genome’s coding regions, specific genomic regions, and epigenetic modifications 
  • Carry out transcriptome profiling, both of coding and non-coding regions 
  • Recognize genes in specific cell types and genetic alterations, such as single nucleotide variants and gene fusions 
  • Complete PCR for next-generation polymerases in the NGS workflow
  • Improve Covid-19 testing.

These progressions aside, developments in NGS will likely see its applications develop, especially as sequencing techniques become more accessible and cost-effective.

Life Sciences Journal BioTechniques

Since releasing its first issue in 1983, the peer-reviewed, open-access journal BioTechniques has attracted a global audience of research professionals who specialize in the life sciences, physics, chemistry, plant and agricultural science, and computer science. These specialists can access an ever-growing collection of high-quality resources, not only in BioTechniques’ journal but also on its website, where users find articles, interviews, eBooks, webinars, podcasts, videos, and infographics that explain the latest in the efficacy of lab methods like western blotting, CRISPR gene editing, polymerase chain reaction, and NGS. As one of Future Science Group’s 34 established journals, BioTechniques sits alongside other reputable journals, such as Future Oncology, Regenerative Medicine, and Nanomedicine.