Key Difference – NGS vs Sanger Sequencing
Next Generation Sequencing (NGS) and Sanger Sequencing are two types of nucleotide sequencing techniques developed over the time. Sanger Sequencing method was widely used for many years and NGS replaced it recently due to its advantages. The key difference between NGS and Sanger Sequencing is that NGS works on the principle of sequencing millions of sequences simultaneously in a rapid way through a sequencing system while the Sanger Sequencing works on the principal of chain termination due to selective incorporation of dideoxynucleotides by DNA polymerase enzyme during the DNA replication and resulting fragment separation by capillary electrophoresis.
CONTENTS
1. Overview and Key Difference
2. What is Nucleotide Sequencing
3. What is NGS
4. What is Sanger Sequencing
5. Side by Side Comparison – NGS vs Sanger Sequencing
6. Summary
What is Nucleotide Sequencing?
Genetic information is stored in the nucleotide sequences of the DNA or RNA of an organism. The process of determining the correct order of nucleotides (using four bases) in a given fragment (in a gene, cluster of genes, chromosome, and complete genome) is known as nucleotide sequencing. It is very important in genomic studies, forensic studies, virology, biological systematic, medical diagnosis, biotechnology and in many other fields to analyse the structure and function of genes. There are different types of sequencing methods developed by scientists. Among them, Sanger sequencing developed by Frederick Sanger in 1977 was widely used and popularized for a long time period until Next Generation Sequencing replaced it.
What is NGS?
Next Generation Sequencing (NGS) is a term used to refer to modern high throughput sequencing processes. It describes a number of different modern sequencing technologies which revolutionized genomic studies and Molecular Biology. Those techniques are Illumina sequencing, Roche 454 sequencing, Ion Proton sequencing and SOLiD (Sequencing by Oligo Ligation Detection) sequencing. NGS systems are quicker and cheaper. Four main DNA sequencing methods are used in NGS systems namely; pyrosequencing, sequencing by synthesis, sequencing by ligation and ion semiconductor sequencing. A large number of DNA or RNA strands (millions of) can be sequenced parallelly. It allows sequencing of the entire genome of organisms within a short time period, unlike Sanger sequencing which takes more time.
NGS has many advantages over conventional sequencing Sanger method. It is a high speed, more accurate and cost-effective process which can be performed with a small sample size. NGS can be used in metagenomic studies, in the detection of variations within an individual genome due to insertions and deletions etc. and in the analysis of gene expressions.
Figure_1: Developments in NGS Sequencing
What is Sanger Sequencing?
Sanger Sequencing is a sequencing method developed by Frederick Sanger and his colleagues in 1977 to determine the precise nucleotide order of a given DNA fragment. It is also known as chain termination sequencing or Dideoxy sequencing. The working principle of this method is the termination of strand synthesis by selective incorporation of a chain terminating dideoxynucleotides (ddNTPs) such as ddGTP, ddCTP, ddATP and ddTTP by DNA polymerase during the replication of DNA. Normal nucleotides have 3’ OH groups for the formation of a phosphodiester bond between adjacent nucleotides to continue the strand formation. However, ddNTPs lack this 3’ OH group and are unable to form phosphodiester bonds between nucleotides. Hence, the chain elongation is ceased.
In this method, the single-stranded DNA to be sequenced serves as the template strand for in vitro DNA synthesis. Other requirements are oligonucleotide primer, deoxynucleotide precursors and DNA polymerase enzyme. When the flanking ends of the target fragment are known, primers can be easily designed for DNA replication. Four separate DNA synthesis reactions are performed in four separate tubes. Each tube has separate ddNTPs, together with other requirements. From the particular nucleotide, a mixture of dNTPs and ddNTPs are added. Likewise, four separate reactions are performed in four tubes with four mixtures. After the reactions, the detection of DNA fragments and conversion of the fragment pattern into sequence information are performed. Resulting DNA fragments are heat denatured and separated by gel electrophoresis. If radioactive nucleotides are used, the banding pattern in the polyacrylamide gel can be visualised by autoradiography. When this method uses the fluorescently tagged dideoxynucleotides, it can be mitigated down the gel read and passed through a beam of laser to be detected by the fluorescent detector. To avoid errors that might arise when a sequence is read by the eye and enter manually into a computer, this method developed into the use of automated sequencer coupled with the computer.
This is the method used to sequence DNA from the Human Genome project. This method is still in use with advanced modifications because it gives accurate sequence information despite being an expensive and slow process.
Figure_2: Sanger Sequencing
What is the difference between NGS and Sanger Sequencing?
NGS vs Sanger Sequencing |
|
| Next Generation Sequencing (NGS) refer to modern high throughput sequencing processes. It describes a number of different modern sequencing technologies | Sanger Sequencing is a sequencing method developed by Frederick Sanger to determine the precise nucleotide order of a given DNA fragment. |
| Cost Effectiveness | |
| NGS is a cheaper process because it reduces time, man power and chemicals. | This is a costly process because it takes time, man power and more chemicals. |
| Speed | |
| This is quicker since both chemical detection and signal detection of many strands are happening parallel. | This is time-consuming since chemical detection and signal detection happens as two separate processes and only on strand can read at a time. |
| Reliability | |
| NGS is reliable. | Sanger sequencing is less reliable |
| Sample Size | |
| NGS requires less amount of DNA. | This method needs a large amount of template DNA. |
| DNA Bases per Sequenced Fragment | |
| The number of DNA bases per sequenced fragment is lower than Sanger method | Generating sequences are lengthier than NGS sequences. |
Summary – NGS vs Sanger Sequencing
NGS and Sanger Sequencing are nucleotide sequencing techniques extensively used in Molecular Biology. Sanger sequencing is an early sequencing method which was replaced by NGS. The main difference between NGS and Sanger Sequencing is that NGS is a high speed, more accurate and cost-effective process than Sanger sequencing. Both techniques created major outbreaks in Genetics and Biotechnology.
Reference:
1. Nowrousian, Minou. “Next-Generation Sequencing Techniques for Eukaryotic Microorganisms: Sequencing-Based Solutions to Biological Problems.” Eukaryotic Cell. American Society for Microbiology, Sept. 2010. Web. 18 Feb. 2017
2. Sanger, F., S. Nicklen, and A. R. Coulson. “DNA sequencing with chain-terminating inhibitors.” Proceedings of the National Academy of Sciences 74.12 (1977): 5463-467. Web.
3. Liu, Lin, Yinhu Li, Siliang Li, Ni Hu, Yimin He, Ray Pong, Danni Lin, Lihua Lu, and Maggie Law. “Comparison of Next-Generation Sequencing Systems.” Journal of Biomedicine and Biotechnology 2012 (2012): 1-11. Web.
Image Courtesy:
“Sanger-sequencing”By Estevezj – Own work (CC BY-SA 3.0) via Commons Wikimedia
“Developments in next generation sequencing” By Nederbragt, Lex (2012) – (CC BY 3.0) via Commons Wikimedia