The massively parallel sequencing technology is known as “next-generation sequencing” (NGS). It provides extremely high throughput, scalability, and speed.
The term “next-generation sequencing” (NGS) refers to high-throughput technologies that can quickly ascertain the sequence of a particular nucleic acid strand, such as DNA or RNA. These tools have changed how we conduct genomics research. “Next-generation sequencing” refers to various technologies that operate according to various operating principles.
The questions scientists can ask and receive answers to have been drastically transformed by next-generation sequencing technology. Innovative data analysis and sample preparation methods offer a wide range of applications. NGS, as an example, enables labs to:
- Sequence whole genomes quickly.
- Deeply sequence the intended areas.
- Quantify mRNAs for gene expression analysis or use RNA sequencing (RNA-Seq) to find novel RNA variations and splicing sites.
- Investigate aspects of epigenetics, including DNA-protein interactions and genome-wide DNA methylation.
- We study rare somatic variants, tumor subclones, and other things by sequencing cancer samples.
- Learn about the human microbiome.
- Discover new pathogens.
Benefits of NGS
Next-generation sequencing is a common technology in functional genomics and can be used to analyze DNA and RNA materials. NGS-based methods provide several advantages over microarray techniques, including:
- It is optional to have prior knowledge of the genome or its properties.
- Due to its single-nucleotide resolution, it is feasible to identify genes connected, alternatively spliced transcripts, allelic gene variations, and single nucleotide polymorphisms.
- Greater signal dynamic range
- Less input DNA/RNA is necessary (nanograms of materials are sufficient)
- More repeatability
Working with next-generation sequencing
- Build a library
From the sample, a sequencing “library” must be built and processed into relatively brief double-stranded fragments from the DNA (or cDNA) sample (100–800 bp). DNA breakage can be carried out in various ways, depending on the particular application, including physical shearing, enzyme digestion, and PCR-based amplification of particular genetic areas. The resultant DNA fragments are subsequently linked to adaptor sequences appropriate to the technology, creating a library of fragments. Additionally, these adaptors might have a special molecular “barcode,” allowing each sample to be marked with a different DNA sequence.
- Amplification of clones
To improve the signal detected from each target during sequencing, the DNA library must be attached to a solid surface and clonally amplified before use. Each unique DNA molecule in the library is bonded to the surface of a bead or flow cell during this procedure, and a set of identical clones is produced by PCR amplification.
- Library of sequences
The entire collection of DNA in the library is sequenced using a sequencing device. Despite the differences across NGS technologies, they all read individual bases as they spread along a polymerized strand using a variation of the “sequencing by synthesis” technique. DNA base synthesis on single-stranded DNA is the first phase in this cycle, which also involves the detection of the integrated base and removing reactants to restart the cycle.
- Data analysis
Each NGS experiment produces a significant amount of detailed data from brief DNA reads. Although each technological platform has its algorithms and data analysis tools, they all employ the same metrics to assess the quality of NGS data sets and have a comparable analytical “pipeline.” Primary, secondary, and final analyses are the three stages of analysis. Check Tekmatic for more information.
With NGS, thousands to millions of short nucleic acid sequences can be rapidly and massively parallelly sequenced. Sequencing vast portions of the genome at a cheaper cost and with higher sensitivity than the traditional sequencing method, such as Sanger sequencing, offers a variety of benefits.
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