What is the Difference Between Sanger and Next Generation Sequencing

The main difference between Sanger sequencing and next-generation sequencing is that Sanger sequencing processes only a single DNA fragment at a time, whereas next-generation sequencing processes millions of fragments simultaneously at a time. Furthermore, Sanger sequencing is analogical while next-generation sequencing is digital, allowing the detection of the novel or rare variants with deep sequencing. Moreover, Sanger sequencing is a fast and cost-effective method for low numbers of targets, generally up to 20 targets, while next-generation sequencing is a time consuming and less cost-effective method. 

Sanger sequencing and next-generation sequencing are the two methods of sequencing the DNA fragments. Moreover, choosing Sanger or next-generation sequencing depends on the benefits and limitations of both methods. 

Key Areas Covered 

1. What is Sanger Sequencing
     – Definition, Process, Importance
2. What is Next Generation Sequencing
     – Definition, Process, Importance
3. What are the Similarities Between Sanger and Next Generation Sequencing
     – Outline of Common Features
4. What is the Difference Between Sanger and Next Generation Sequencing
      – Comparison of Key Differences

Key Terms 

Next Generation Sequencing (NGS), Parallel Sequencing, Sanger Sequencing (SGS), Sequencing, Sequencing Depth

What is Sanger Sequencing 

Sanger sequencing (SGS) is the first generation sequencing method developed by Fredric Sanger in 1977. It involves the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication. Then, the producing amplicons are separated by capillary electrophoresis. Generally, Sanger sequencing serves as a fast and cost-effective sequencing method for small-scale projects with less than 100 amplicon targets. Moreover, it is better for the sequencing of single genes.

Figure 1: Sanger Sequencing

Furthermore, Sanger sequencing is an analogical method which generates a single sequence by combining signals from all DNA fragments in the sample. It does not allow the isolation of individual signals. Thus, the resultant signal is a mixed-signal, which does not allow the identification of variants, which occur below 25% frequency in a sample. 

What is Next Generation Sequencing 

Next-generation sequencing (NGS) is a second-generation sequencing method. Moreover, it is a high-throughput DNA sequencing approach with the concept of massively parallel processing. Genome Analyzer/HiSeq/MiSeq (Illumina Solexa),  SOLiD System (Thermo Fisher Scientific), Ion PGM/Ion Proton (Thermo Fisher Scientific), and HeliScope Sequencer (Helicos BioSciences) are the several platforms currently performing next-generation sequencing. Generally, they can sequence 1 million to 43 billion short reads (50-400 bases each) per instrument run. 

Figure 2: Clonal Amplification in Illumina Sequencing

Moreover, the main feature of next-generation sequencing is that it can perform a parallel investigation of multiple targets.  It has increased speed and efficiency of mutation detection. Generally, in somatic cancer mutations, tumors are heterogeneous and contain both cancer cells as well as the normal cells. However, the preparation of a DNA library by clonal amplification in next-generation sequencing for parallel sequencing helps to physically separate signals originating from each target DNA molecule in the library. Therefore, this allows the separation of DNA sequences of cancer cells from the DNA sequences of normal cells. In overall, the next-generation sequencing is a digital sequencing method with a higher depth of coverage variants.  

Similarities Between Sanger and Next Generation Sequencing 

• Sanger and next-generation sequencing are the two main methods used to determine the nucleotide sequence of DNA fragments. 
• Their technology is similar and involves the addition of fluorescent nucleotides onto the growing template strand by DNA polymerase. 
• Moreover, the identification of added nucleotides is by their fluorescent tag. 
• Also, both are automated techniques.  

Difference Between Sanger and Next Generation Sequencing 

Definition 

Sanger sequencing refers to a low-throughput method used to determine a portion of the nucleotide sequence of an individual’s genome, while the next-generation sequencing refers to a high-throughput method used to determine a portion of the nucleotide sequence of an individual’s genome. Thus, this is the main difference between Sanger and next generation sequencing.

Other Names 

The other names for Sanger sequencing are dideoxy chain termination method or capillary electrophoresis sequencing, while the other names for next-generation sequencing are massive parallel sequencing or massively parallel sequencing.  

Generation 

Sanger sequencing is a first-generation sequencing method, while the next-generation sequencing is a second-generation sequencing method. 

Commercialization 

Moreover, Sanger sequencing was first commercialized by Applied Biosystems, while the dominant next-generation sequencing platform is Illumina.  

Size of DNA Fragments 

Another difference between Sanger and next generation sequencing is that while Sanger sequencing works better for 750-1,000 base pair fragments, next-generation sequencing works better for around 20 million base pairs long fragments.  

Number of Samples at a Time 

Furthermore, Sanger sequencing can only process a single DNA fragment at a time while next-generation sequencing processes millions of fragments simultaneously at a time. 

Depth of Coverage 

Importantly, the Sanger sequencing is analogical as it combines all DNA fragments in a mixture to produce a single sequence, while the next-generation sequencing is digital as it allows to separate each individual data coming from a single molecule in the mixture.  

Sensitivity 

Also, sensitivity is another difference between Sanger and next generation sequencing. Sanger sequencing is a less sensitive method with a limit of detection around 15-20% while next-generation sequencing is a highly sensitive method with a limit of detection is less than 1%. 

Cost per Low Number of Samples 

Besides, Sanger sequencing is fast and cost-effective up to 20 samples, while the next-generation sequencing is time-consuming and less cost-effective up to 20 samples. 

Cost per a Higher Number of Samples 

Sanger sequencing is less cost-effective for a higher number of samples, while next-generation sequencing is cost-effective for a higher number of samples. 

Clinical Research Sequencing 

One other difference between Sanger and next generation sequencing is that the Sanger sequencing is the ‘gold standard’ for clinical research sequencing, while the next-generation sequencing is becoming common in clinical labs. 

Applications 

Moreover, the Sanger sequencing is important for fragment analysis, microbial identification, STR analysis, NGS confirmation, etc., while the next-generation sequencing is important for genome sequencing including pathogenic microbial genomes, transcriptome analysis, mutation detection, etc. 

Conclusion 

Sanger sequencing is the first generation sequencing method, which involves the amplification of a target DNA fragment with fluorescent-labelled dideoxynucleotides and analysis through capillary electrophoresis. Generally, this method is fast and cost-effective for a small number of samples. As it is an analogical method, it has less sensitivity. On the other hand, next-generation sequencing is the second generation sequencing method with similar technology to Sanger sequencing. It is a massively parallel sequencing method that processes millions of samples at once. Furthermore, the main feature of next-generation sequencing is its sequencing depth, which allows the detection of variants. Therefore, the main difference between Sanger and next generation sequencing is the number of samples processing and the depth of sequencing.  

References:

1. Arsenic, Ruza et al. “Comparison of targeted next-generation sequencing and Sanger sequencing for the detection of PIK3CA mutations in breast cancer.” BMC clinical pathology vol. 15 20. 18 Nov. 2015, doi:10.1186/s12907-015-0020-6

Image Courtesy:

1. “Sanger-sequencing” By Estevezj – Own work (CC BY-SA 3.0) via Commons Wikimedia   
2. “Cluster Generation” By DMLapato – Own work (CC BY-SA 4.0) via Commons Wikimedia