Next-Generation Sequencing (NGS) is a revolutionary technology for the world of genomics and molecular biology. Its ability to process entire genome sequences has enabled scientists to detect mutations on a much wider scale, in comparison to the traditional sequencing method, Sanger sequencing.
The application of NGS varies with multiple uses within the fields of clinical oncology, microbiology, haematology and pathology. The fast sequencing of millions of DNA and RNA molecules simultaneously through NGS technology methods, has provided researchers with new findings, therefore resulting in key breakthroughs for genetics and biological systems.
Figure 1. Schematic of a Next Generation Sequencing (NGS) workflow
For a detailed explanation of each step, see below:
Figure 2. DNA extraction from sample
A Next-Generation Sequencing workflow starts with isolating the nucleic acids from various sample types. These include bulk tissue, individual cells or biofluids. RNA samples are made into complementary DNA (CDNA).
Once extraction and purification are completed, a Quality Control (QC) step is required to check the integrity and quantity of the sample before further processing.
Figure 3. DNA fragmentation to create a new sequencing library
After QC is completed, library preparation is a crucial stage of NGS to ensure the compatibility of DNA or RNA samples with the sequencer. The genetic sample of DNA is fragmented into smaller pieces and specific adaptors are attached to both ends of each fragment, thereby creating a sequencing library.
Learn more about OGT’s library preparation workflow
Figure 4. Synthesis by Sequencing
When the sample libraries are loaded into the sequencer, the adaptors on the fragments bind to the flow cell. The libraries are then up sampled to form dense clusters of single-stranded DNA fragments. This increases the resolution of data that can be achieved in the next step of synthesis. The new library can be up sampled where the quality of data is improved, and the sample material is given a higher resolution or can become denser.
The cluster generation process involves a process known as bridge amplification, a type of polymerase chain reaction (PCR) and amplifies DNA bound fragments into clonal clusters, producing millions of single-stranded DNA copies.
Once amplified and clustered, the library fragments are then used as a template for fluorescently labelled nucleotides to synthesise a complementary strand on each fragment. Each time a fluorescently labelled nucleotide is added, a light signal is emitted that will be different for each type of nucleotide. These light signals from the DNA clusters are recorded. The wavelength and intensity of light emission are used to identify the sequence of the templates.
Figure 5. Bioinformatic analysis
Once sequencing is complete, data analysis is a crucial phase where researchers can identify the genetic mutations or variations, and extract meaningful biological insights from the NGS data. With efficient data handling and accurate interpretation, these NGS features are vital for applications in genomics and personalised medicine.
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