Question

Describe the methodology behind next-generation sequencing techniques.

Short answer

The methodology behind the next-generation sequencing is pyrosequencing, massive parallel pyrosequencing, and emulsion PCR.

Step-by-step solution

Introduction

NGS stands for next-generation sequencing, which is a massively parallel sequencing technology that provides ultra-high throughput, scalability, and speed. The method is used to determine the nucleotide order of entire genomes or specific DNA or RNA portions.

Methodology behind Next Generation Sequencing

Pyrosequencing: The first after the primer is attached and duplicated by DNA polymerase, in which the deoxyribonucleotide addition is visible due to the release of pyrophosphate. The sulfurylase enzyme can convert this to chemiluminescence.

Massive parallel pyrosequencing: The first step in high-throughput pyrosequencing is to break down genomic DNA into fragments, which are then ligated to a pair of adaptors.

The DNA fragments can be linked to small metabolic beads using these adaptors. The 5'end is biotin-labeled, and the beads are streptavidin-coated; biotin has a strong affinity for streptavidin. Because only one fragment is linked to each bead, each fragment can now be magnified.

These attachments now serve as an annealing station for the primers. PCR is used for amplification, and copies are created for sequencing. This means that the same pair of primers can be used for all of the fragments. Even the fragments themselves contain a variety of sequences.

If you do another PCR right away, you'll get a combination.

The next stage is to conquer it.

Emulsion PCR: To resolve this issue, Emulsion PCR has been completed. It's a type of DNA amplification in which a single DNA molecule is isolated in microreactors using water and oil. Each droplet includes all of the chemicals and is also segregated from other droplets due to the oil.

Following PCR, aqueous droplets are placed in wells on a plastic strip, one droplet and one PCR result in each well. In each well, pyrosequencing is performed.

Interpretation

Thus, the demand for cheaper and quicker sequencing after the first human genome sequence results in second-generation sequencing, also known as next-generation sequencing. All methods need library preparation; but, in this case, the library preparation is the more streamlined line; each sample can be a population, and no clonal purification is required. It's good for clone checking and sequencing amplicons.