Next Generation DNA Sequencing

A sequence tells a scientist the genetic information carried on a particular DNA or RNA segment. For example, the sequence can be used to determine where genes are located on a DNA strand and where regulatory instructions turn those genes on or off. 

In the mid-90s, colleges started teaching their undergraduates about DNA sequencing, with DNA sample amplification tech as the new kid on the block. The Human Genome project was ongoing, and the first human sequence had yet to be completed. 

Twenty-five years later, DNA sequencing is done regularly for many and has helped dramatically with medical and forensic needs. We are now entering a whole new era of sequencing that is the “next generation.” Let’s look at this change and how this generation alters science and medicine.

What Is Next Generation DNA Sequencing?

Next generation DNA sequencing (NGS) started gaining notoriety in the early 2010s and is a term that describes DNA sequencing technologies that have revolutionized genomic research.  

Original DNA Sequencing

To understand NGS, we need to understand the original type of DNA sequencing. 

First, a DNA strand was copied to create enough material. Then one by one, the base pairs were determined using gels with capillaries that pulled them through using electricity, the chain-termination method, or as it is commonly known, Sanger sequencing. 

The Human Genome Project used Sanger sequencing, which multiple international teams utilized to decipher the human genome, taking 13 years and $3 billion to produce the final draft released in 2003. By 2008, using several NGS techniques, the famous discoverer of DNA, James Watson’s genome sequence, was provided to him on a hard drive at an estimated cost of $1 million. 

In 2011, Apple co-founder and billionaire Steve Jobs had his DNA sequence done to help in his cancer fight for $100,000. Using NGS, a lab can now sequence an entire human genome in only one day at the cost of $100 (Ultima Genomics).  

How NGS Works

NGS involves a stepwise process (four total steps) that breaks up the sample (DNA or RNA) and sequences the parts simultaneously to get faster results.

Source: Illumina

The process is generally as follows:

1. Sample preparation involves fragmenting DNA/RNA into multiple pieces (millions for the human genome) and then adding “adapters” to the ends of the DNA fragments.

2. Cluster generation is where the separated strands are copied millions of times to produce a larger sample. 

3. Sequencing the libraries: each of the strands is sequenced with unique fluorescent markers.

4. A genomic sequence is formed by reassembling the strands using data analysis techniques. 

In principle, the NGS concept is similar to capillary electrophoresis (gels used to sequence DNA in Sanger sequencing). The critical difference is that with NGS, because the fragments are broken up, the sequences of millions of fragments are obtained in a massively parallel fashion, improving accuracy and speed while reducing the cost of sequencing.  

NGS’ Impact

Compared to the conventional Sanger sequencing method’s capillary electrophoresis, NGS’ short-read massively parallel sequencing technique is a fundamentally different approach that revolutionizes our sequencing capabilities, launching the second generation of sequencing methods.

NGS allows for the sequencing of both DNA and RNA at a drastically cheaper cost  than Sanger sequencing, and it, therefore, has revolutionized the studies of genomics and molecular biology. 

NGS’ Advantages

Because NGS can analyze both DNA and RNA samples, it’s a popular tool for functional genomics. In addition, NGS has several advantages over microarray methods.

· A priori knowledge of the genome or of any genomic features is not a requirement.  

· NGS offers single nucleotide resolution, which detects related genes and features, genetic variations, and even single base pair differences. In short, it can spot slight differences in code between two samples. 

· NGS has a higher dynamic signal range, making it easier to read.

· NGS requires less DNA or RNA as an input (nanograms of material are sufficient). 

· NGS has higher reproducibility. Because of its other advantages, the chance of an error between repeated tests is reduced.  

Most Common NGS Technologies

Three sequencing methods are and were widely used that fall under the NGS umbrella:

· Roche 454 sequencing (discontinued in 2016). This method uses a pyrosequencing technique that detects a pyrophosphate release. It uses bioluminescence (a natural light signal). Broken-up DNA stands had unique markers attached.  


· Illumina (Solexa) sequencing. The Illumina process simultaneously identifies DNA base pairs. This is done as each base emits a different and unique fluorescent signal, continuously added to the nucleic acid chain.


· Ion Torrent (Proton/PGM) sequencing. This kind of sequencing measures the direct release of positive Hydrogen protons when incorporating individual base pairs. They are released when added by a DNA polymerase. The Ion Torrent method differs from the previous two methods because it is not using a light measurement to do the sequencing.  


How Is NGS Being Used?

The advent of NGS has changed the biotechnology industry. There are now new questions that scientists can ask and get the answers to that were either cost-prohibitive or the samples needed were more significant than the available material. The main applications possible with NGS include:

· Rapidly sequencing the whole genome of any life form, from prions and RNA viruses to individual humans and other mammals. 

· Utilize RNA sequencing to discover novel RNA variants and splice sites.

· Quantify mRNAs for gene expression.

· Sequence cancer samples to study rare variants, specific tumor subtypes, and more.

· Identify novel pathogens (such as viruses in bats).

What Can NGS Do? 

Notable organizations, such as Illumina, 454 Life Sciences, Pacific Biosciences, and Oxford Technologies Nanopore, are working on getting prices down so nearly anyone can get sequencing done. For example, Ultima Genomics has claimed a cost of $100 for its sequencing. Now, companies are marketing benchtop sequencing platforms that will bring these advances to as many labs as possible.  

Source: Illumina

The Illumina NextSeq Sequencer (above) is a benchtop system that can do nearly any task except “Large Whole-Genome Sequencing.” However, there is a cost of $210,000-335,000.  

We expect NGS to become more efficient and affordable over time, and these cost reductions will revolutionize several genomics-related fields. Currently, all NGS approaches demand “library preparation” after the DNA fragmentation step, where adapters are attached to the ends of the various fragments. That is generally followed by a DNA amplification step to create a library that can be sequenced with the NGS device. 

As we know more about different DNA molecules, we can develop ways to fight disease through gene therapy or particular drugs. This knowledge will help change our way of thinking about medicine.  

Third Generation Sequencing

A new class of sequencing tech, called third-generation sequencing or TGS, is being developed. These technologies can sequence single DNA molecules without the amplification step, producing longer reads than NGS. 

Single-molecule sequencing was started in 2009 by Helicos Biosciences. Unfortunately, it was slow and expensive, and the company went out of business in 2012. Nonetheless, other companies saw the benefit and took over the third-gen space.  

Pacific Bioscience has its “Single-Molecule Sequencing in Real Time (SMRT),” and Oxford Nanopore has nanopore sequencing. Each can produce long reads of 15,000 bases from a single DNA or RNA molecule. This evolution means smaller genomes can be produced without the biases or errors inherent to amplification. 

Closing Thoughts

The DNA sequence is a simple format in which a broad range of biological marvels can be projected for high-value data collection. Over the past decade, NGS platforms have become widely available, with the costs of services lowering by orders of magnitude, much faster than Moore’s law, democratizing genomics, and putting the tech into the hands of more scientists. 

Third generation sequencing will require robust protocols and practical data approaches. The coming expanse of DNA sequencing will require a complete rethinking of experimental design. Still, it will accelerate biological and biomedical research, enabling the analysis of complex systems inexpensively and at scale. We can then fight and prevent genetic diseases before they become realized issues.

Disclaimer: The information provided in this article is solely the author’s opinion and not investment advice – it is provided for educational purposes only. Using this, you agree that the information does not constitute investment or financial instructions. Do research and reach out to financial advisors before making any investment decisions.

The author of this text, Jean Chalopin, is a global business leader with a background encompassing banking, biotech, and entertainment. Mr. Chalopin is Chairman of Deltec International Group,

The co-author of this text, Robin Trehan, has a bachelor’s degree in economics, a master’s in international business and finance, and an MBA in electronic business. Mr. Trehan is a Senior VP at Deltec International Group,

The views, thoughts, and opinions expressed in this text are solely the authors’ views, and do not necessarily reflect those of Deltec International Group, its subsidiaries, and/or its employees.