Next-Generation Sequencing vs. Sanger Sequencing: What's the Difference? - HealthSurgeon - Healthy Living and Fitness

DNA sequencing involves identifying the order of nucleotide bases, including Thymine, Guanine, Cytosine, and Adenine, encoding the exact genomic information. The technology involved in DNA sequencing developed from its inception, with Frederick Sanger’s work, which sequenced fully based on the plus & minus method. Ultimately, the Sanger dideoxy method or chain termination provided the most important foundation for the growth of DNA sequencing and a good example we see in NGS (next-generation sequencing). So, the question is – what is the difference between the two?

The NGS

It is a massive technology with approaches, which are characterized by carrying out many sequencing reactions. With NGS technology development, whole genome sequencing is more affordable and faster. The NGS processes are liable for the power and speed delivered by today’s approaches to sequencing. First, the library of nucleic acid, which is being analyzed gets generated. If you are using RNA as the sample, then it is converted into cDNA and used to create a library. Afterwards, DNA strands get broken and short adaptors are then ligated onto the fragment’s end. The adapters may serve many functions, including providing a unique barcode, which allows pooling of permits. Lastly, adapters act as primer binding sites to amplify during the reaction.

Sanger Sequencing

This is the first-generation sequencing that Frederick Sanger and his team developed in the 1970s. It is a technique, which uses dideoxynucleotides to terminate replication of DNA, leading to fragments of DNA of various lengths. The fragments’ lengths are dictated by gel electrophoresis, and DNA sequences are read from the gel. Sanger sequencing is an accurate technique for DNA sequencing, but it is a slower method and sequences short fragments. But still, the technique is a gold standard that helps to sequence small DNA regions with more accuracy. Nucleotide pools that are used to grow new chains of DNA have a mixture of unlabeled bases. Moreover, the labels on the bases of the terminator all have different color fluorescent tags to ensure every region is copied. When polymerase encounters dideoxynucleotides, it won’t add extra nucleotides to the DNA sequence. This, in turn, generates fragments, which ‘terminate’ at all the positions in the sequences getting copied. The process of chromatography helps to separate those fragments in the sequencing instrument.

Differences

In terms of principle, the idea behind NGS and Sanger sequencing are the same. In both techniques, DNA polymerase adds fluorescent nucleotides one at a time onto the growing templates. All the incorporated DNA nucleotides are identified by their fluorescent tags. However, the key difference between the two methods is seen in the sequencing volume. Although the Sanger technique only sequences single DNA fragments at a time, next-generation sequencing is parallel massively – meaning it sequences numerous fragments at the same time on every run. The process translates into sequencing thousands of genes. In addition, next-generation sequencing provides more discovery power to help detect rare or novel variants with deeper sequencing. Other differences include:

1. Throughput & Scalability

The NGS’s hallmark lies in the technique’s notable scalability and throughput. It has the capability of sequencing numerous DNA fragments in just one operational cycle; thus, facilitating concurrent analysis and extensive genomic coverage, which spans thousands of gene regions or genes. This attribute outpaces the constrained throughput inherent in the first-generation sequencing technology that traditionally processes fragments of DNA serially.

2. Operation Principle

The first-generation sequencing works by fluorescently integrating tagged ddNTPs (dideoxynucleotides) during the synthesis of DNA. Each dideoxynucleotide stops the elongation of DNA strands at a precise nucleotide location, thereby facilitating the determination of DNA sequence through capillary electrophoresis. On the other hand, next-generation sequencing uses different kinds of mechanisms, including nanopore-based sequencing, single-molecule sequencing, and reversible terminator chemistry, to achieve high-throughput sequencing.

3. Sensitivity & Detection Limit

Next-generation sequencing provides superior sensitivity when compared to first-generation sequencing, especially when it comes to detecting rare mutations as well as low-frequency variants. The deep capacity of sequencing in NGS helps to identify subtle genetic variations, which are present at frequencies. On the other hand, the first-generation sequencing might have lower sensitivity, with a limit of detection. The detection limit is between 15% and 20%.

In summary, both methods of sequencing delve into the analysis of DNA at the level of single-base, but their approaches significantly vary. The first-generation sequencing depends on the pioneering determination method that Frederick Sanger introduced. The technique generates DNA fragments of target sequences, all initiated from primers but terminated at different lengths. NGS, on the other hand, involves a wide range of methods for sequencing libraries of DNA. These libraries consist of short DNA fragments’ collections, sourced from genomic RNA or DNA. But in terms of accuracy and throughput, NGS is better than Sanger sequencing.