Next Generation Sequencing (NGS), also known as “massive parallel sequencing” is an overarching term that covers several high throughput approaches to DNA sequencing. It is a popular method as it offers ultra-high throughput, scalability and speed compared to traditional Sanger sequencing. The technology is used to determine the order of nucleotides in an entire genome or targeted regions of DNA. This information can be used for multiple purposes such as identifying mutations or copy number variations.
Developed in the 1990s and commercially available since 2005, these technologies involve the simultaneous sequencing of millions of short fragments. Once these short sequences have been determined, bioinformatic software is used to combine and “map” these sequences to create a larger sequence, usually an entire genome.
How does NGS work?
1. The first step in NGS methods is to ensure that the DNA (or RNA) is fragmented. This step is not necessary for NIPT as cell free circulating DNA is already fragmented.
2. Specialised adapters (short DNA sequences) are added to each end of the fragments of DNA so that they can be identified and tracked.
3. DNA fragments with attached adapters are then amplified to create “DNA libraries” for each sample. The adapters attached in this step are unique for each sample so that multiple samples can be sequenced at once and still be assigned to a patient in later steps.
4. These amplified DNA libraries may be purified to concentrate DNA fragments of interest.
5. DNA libraries are sequenced using a specialist instrument (Illumina NextSeq550Dx for IONA® Nx).
- Libraries are loaded on to a “flow cell” on the instrument where clusters of DNA fragments are amplified in a process known as cluster generation. This results in millions of copies of single stranded DNA on the flow cell.
- Once cluster generation is complete, a process known as sequencing by synthesis takes place. Here free nucleotides bind to the DNA fragments created during cluster generation and generate a fluorescent signal that is picked up by the instrument, identifying what each base added to the fragment is.
- One base at a time, this method is used to determine the sequence of DNA fragments.
- This method is also known as massive parallel sequencing as these steps happen with millions of fragments simultaneously.
6. Sequences of these DNA fragments determined during sequencing are reassembled to form a larger sequence, allowing fragments to be mapped to a genome.
7. Data generated allows the order of nucleotides in an entire genome or targeted region to be determined.