NEXT-GENERATION SEQUENCING

Shivani Vaja, former student President Science College

DNA Sequencing is the systematic process of determining, performing, and analyzing the sequence of nucleotides ( the order of adenine, guanine, cytosine, and thymine nucleotides ) in DNA. The technique of resolving DNA sequence was known as Sanger sequencing. This methodology included the separation of fluorescently labeled DNA fragments based on their length on a polyacrylamide gel (PAGE). To visualize and identify dye was used. This method required a high number of samples, which made it more time-consuming and costly. Time and money became a constraint, reducing the efficiency of sequencing and posing obstacles to researchers.

By that time, researchers had been working on improving DNA sequencing methods and devising new ones, such as Maxam-Gilbert sequencing, Chain-termination method, and Shotgun sequencing. However, these approaches were insufficient for huge genomes, and as a result, the intriguing discovery of next-generation sequencing, also known as Massive parallel sequencing or second-generation sequencing, took place.

The phrase “next-generation sequencing” (NGS) catches all term that is used to describe various modern sequencing technologies. Next-Generation Sequencing (NGS) is also known as the high-throughput sequencing method. The said technologies allowed sequencing of DNA and RNA faster and cheaper than the other DNA Sequencing methods. In comparison to Sanger sequencing, NGS technologies are exceedingly capable, allowing massive parallel processing and extraordinarily high throughput from numerous samples. An entire human genome can be sequenced in a single day using NGS, whereas it took 20 years to complete a human genome project using older methods.

NGS Workflow

Next-generation methods of DNA sequencing are generally performed by following steps.

(1) Library preparation: To prepare the library, the DNA sample is fragmented into small strands using enzymes, and adaptors are ligated to these fragments with the help of DNA ligase.

The library fragments must be grouped in PCR colonies that contain numerous copies of a given library fragment for sequencing to be successful. Due to the planar attachment of these colonies, the array’s characteristics can be manipulated enzymatically in parallel.

(2) Amplification:  Amplification of Library is an important step as it becomes a mandate to get desire signals from the sequencer that needs to be strong enough for easy detection. The Amplification without authentication often results in preferential amplification for some specific library fragments.

Instead, amplification processes that use PCR to produce vast numbers of DNA clusters are available.

• Emulsion PCR: Emulsion oil, beads, PCR mix, and library DNA are combined to generate an emulsion that forms microwells. Polymerase amplifies the reverse strand as it anneals to the bead. The opposite strand denatures and is liberated from the bead, only to re-anneal to the beads, resulting in two distinct strands. The process is then repeated 30-60 times, resulting in DNA clusters. This approach is not widely accepted for being time-consuming due to the several processes it entails. 

• Bridge PCR: Primers that are complementary to the primers connected to the DNA library fragments are extensively deposited on the flow cell’s surface. The DNA is then randomly linked to the cell’s surface and exposed to reagents for a polymerase-based extension. The free ends of single strands of DNA bind to the cell surface via complementary primers when nucleotides and enzymes are added, forming bridging structures. Enzymes then interact with the bridges to make them double-stranded, resulting in two single-stranded DNA fragments clinging to the surface nearby when denaturation occurs. This procedure is repeated several times, resulting in clonal clusters of localized identical strands. To avoid overcrowding and optimize cluster density, reagent concentrations must be continuously maintained.

(3) Sequencing Using Different Methods: In the last step of DNA sequencing different and appropriate methods are put to use to get the proper outcome of an experiment.  

 Types of Next Generation Sequencing (NGS)

• Illumina (Solexa Sequencing): Illumina or Solexa Sequencing works by simultaneous recognition of DNA Bases. It generates a unique fluorescent signal in each base and combines them into a nucleic acid chain. Deep sampling and uniform coverage are used to generate a consensus, assuring the highest determination of genetic differences. It utilizes primarily different approaches from the Sanger chain-termination method. It influences sequences by synthesis technique, tracking the addition of labeled nucleotides as DNA chain is copied in the parallel term. Systems can be deployed from the output of 300 kilobases up to multiple terabases in one run, mostly depends on instrument type, performance, and configuration. 
• Roche (454 Pyrosequencing): Roche Sequencing is based on pyrosequencing, a method which detects pyrophosphate release, again using fluorescence, after nucleotides are established by polymerase to a new stand DNA. 

Instead of using dideoxynucleosides to prevent chain amplification, trace the production of pyrophosphate when nucleotides are mixed up with the DNA chain. Generic adaptors are added to the bottom, which is strengthened to beads, basically, one DNA fragment per bead, further amplified by using specific primers.

Each bead is placed in a single well of a slide so that each well has a single bead. The wells also cover DNA polymers, followed by a sequencing buffer. The four separate dNTPs are then progressively made to flow in and out of the wells over the colonies after an ssDNA sequencing primer hybridizes to the end of the strand (primer-binding region). 

Pyrophosphate is produced when the right dNTP is enzymatically integrated into the strand. The pyrophosphate is converted to ATP in the presence of ATP sulfurylase and adenosine.

This ATP molecule is required for the luciferase-catalyzed conversion of luciferin to oxyluciferin, which generates light detectable by a camera. The amount of base injected determines the relative intensity of light.

In addition to these two, Sequencing by ligation (SOLiD) and Ion torrent semiconductor sequencing is also used in numerous applications including inherited diseases, oncology, infectious diseases, reproduction genomics, and much more.   

Benefits of using NGS 

As a next-generation sequencing method, it is used to analyze, test, and measure the performance of RNA and DNA samples which is a very popular tool in functional genomics. The Following major benefits make the NGS better in comparison to other methods of the same streamline.

• Basic or prior knowledge of the genomic features is not mandatory in the case of DNA NGS  Methods. 
• It allows the utilization of RNA sequencing to discover novel RNA variants.
• It provides a high dynamic range of signals. 
• Since it requires less DNA/RNA as input, the cost is automatically reduced.
• It is also available for higher production.
• Capability to sequence hundreds and thousands of genes or gene regions instantaneously. 
• Lower time to detect early errors and their genuine solution.
• It allows researchers to discover new infections.
• Unlike other sequencing methods, the NGS method provides single-nucleotide resolution. So, it is possible to trace related genes, allelic gene variants, and single nucleotide polymorphism.

Also read: Chernobyl tree frogs show no effect of radiation in their blood

Source :

1. Mardis, E. R. (2008). Next-generation DNA sequencing methods. Annu. Rev. Genomics Hum. Genet., 9, 387-402. https://doi.org/10.1146/annurev.genom.9.081307.164359
2. Kulski, J. K. (2016). Next-generation sequencing—an overview of the history, tools, and “omic” applications. Next generation sequencing-advances, applications and challenges, 3-60. http://dx.doi.org/10.5772/61964
3. Ansorge, W. J. (2016). Journal of Next Generation Sequencing & Applications. http://dx.doi.org/10.4172/2469-9853.S1-005

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