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NGS-Different-Sanger-Sequencing

How is NGS Different from Sanger Sequencing?

Introduction

Sequencing DNA involves biochemical processes to ascertain the order (sequence) of nucleotides of DNA strands. Maxam-Gilbert and Sanger methods were developed over 40 years ago, but the Sanger method soon dominated the DNA sequencing arena. With this method, DNA is repeatedly copied resulting in DNA fragments of varying lengths. The ends of each fragment are labeled with a chain terminator, and this allows the determination of the DNA sequence. This approach has led to many important discoveries over the years. However, this approach has proved quite expensive and not adaptable for large-scale sequencing of large genomes. Recent years have afforded the ability to overcome these limitations with the development of next-generation sequencing (NGS). This is a massive parallel sequencing technology that provide a means to perform large-scale sequencing tasks with incredibly increased speed and cost-effectiveness.

History and Advancement of DNA Sequencing 

The Sanger method is based on chain termination. A specific dideoxynucleotide is used to terminate the elongation reaction of DNA. Four different DNA elongation reactions with reaction-starting primers are needed, each containing one of four dideoxynucleotides (representing the four normal DNA nucleotides, and each with a different color dye label). Since dideoxynucleotides lack the 3’ hydroxyl group needed for bonds to form between two nucleotides, DNA elongation stops where a dideoxynucleotide is incorporated.

Sanger sequencing started with the use of radiolabel and sequencing gels. This progressed to the separation of the resulting DNA fragments of different lengths by capillary gel electrophoresis and detection by a laser in the electrophoresis apparatus. Peaks are generated in a chromatogram, which are read to determine the DNA sequence. Next-generation sequencing is becoming a gold-standard in sequencing efforts with Sanger sequencing used to verify NGS data, particularly in basic research efforts where new discoveries are made using NGS.

The Next-Generation Sequencing Era

Next-generation sequencing is a massive parallel high-throughput sequencing technology. There are now a variety of NGS techniques that take advantage of different technological approaches. However, all NGS approaches are characterized by the conduct of numerous (millions) concurrent sequencing reactions (parallel reactions). This approach provides the speed of obtaining large amounts of sequencing information, and on a microscale. Given these characteristics, the cost to sequence a genome is significantly lower than that using Sanger sequencing. About 20 years ago, initial draft human genome sequencing with the Sanger approach cost about $300 million (1). Now, with NGS, the cost is under $1500.

Technical Overview

The technical processes of NGS in general are responsible for the speed and power of the sequencing approach. First, a DNA (or RNA) library is created. This is done by breaking up the DNA strands and ligating DNA adapters known sequence that anchor the DNA to a support (bead, glass plate). The created library is amplified before the actual sequencing phase. The library is the template used to make new fragments of DNA via numerous PCR reactions.

Using the single nucleotide system as an example, the immobilized DNA is mixed at each cycle of sequencing with a solution containing one nucleotide, which is incorporated where it is complementary to the sequence. The surface or support with the immobilized DNA is washed and another solution with a different nucleotide is used. The incorporation of the nucleotides is detected by various techniques depending on the sequencing system used.

For instance, reversible terminator sequencing, uses a reversible terminator with a fluorescent label that is imaged when each nucleotide is added. This is then cleaved to allow incorporation of the next nucleotide resulting in base-by-base sequencing that is highly accurate. Adapter sequence information is removed, and the sequence data is analyzed using a genome reference and bioinformatics methods. New, effective, and more easily assessable sequence analysis methods are ongoing (2).

Benefit to Research and Clinical Medicine Advancement

The use of NGS has rapidly provided biomedical advances applied to a diversity of bioscience fields. This includes, but not limited to, the use of this information in personalized medicine, epidemiology, forensics, and basic science for the discovery of new genes. Using NGS to analyze RNA now allows accurate and high-throughput analysis of a transcriptome. The massive amounts of NGS sequencing data to date has contributed to the ability to apply sequencing information in a timely manner to facilitate diagnostic efforts for many disease including very rare genetic disorders.

Conclusions

Rapid and inexpensive NGS sequencing technologies are providing a means to perform routine whole genome and exome sequencing that provides a record amount of information at extremely reduced costs when compared to Sanger sequencing. This technology allows its use for diagnostic efforts that were previous cost-prohibitive and too slow to be effectively actionable for patients. Now, the information can be obtained in days instead of years and applied to not only diagnostic endeavors, but to tailor treatment to an individual based on information regarding genetic variants that provide clues to a person’s likelihood to successfully respond to a therapeutic approach.

 

References

  1. National Human Genome Research Institute. The Cost of Sequencing a Human Genome. Available at: https://www.genome.gov/27565109/the-cost-of-sequencing-a-human-genome/  Last Updated: July 6, 2016
  2. Souilmi Y, Lancaster AK, Jung JY, Rizzo E, Hawkins JB, Powles R, Amzazi S, Ghazal H, Tonellato PJ, Wall DP. Scalable and cost-effective NGS genotyping in the cloud. BMC Med Genomics. 2015 Oct 15;8:64.

 

 

 

About the Author:

Dr. Stacy Matthews Branch is a biomedical consultant, medical writer, and veterinary medical doctor. She owns Djehuty Biomed Consulting and has published research articles and book chapters in the areas of molecular, developmental, reproductive, forensic, and clinical toxicology. Dr. Matthews Branch received her DVM from Tuskegee University and her PhD from North Carolina State University.
Dr. Branch’s Profile 

Written by Macrogen Corp.

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