Next-generation sequencing (NGS) is a massively parallel sequencing technology that offers ultra-high throughput, scalability, and speed. The technology is used to determine the order of nucleotides in entire genomes or targeted regions of DNA or RNA.
4 steps of next-generation sequencing
(B) cluster generation,
(C) sequencing, and
(D) alignment and data analysis.
(A)Library Preparation :
Library preparation is the first step of next generation sequencing. It allows DNA or RNA to adhere to the sequencing flowcell and allows the sample to be identified. Two common methods of library preparation are ligation-based library prep and tagmentation-based library prep.
(B) Cluster Generation:
There are two short DNA sequences attached to the flow cell surface, (here in green and yellow). Each of these short DNA molecules is complementary to one strand of the adapter previously added to the DNA sample, thus enabling a specific binding on the flow cell.
DNA sequencing is the process of determining the sequence of nucleotides (As, Ts, Cs, and Gs) in a piece of DNA. In Sanger sequencing, the target DNA is copied many times, making fragments of different lengths.
(D) Alignment and Data Analysis :
First, the DNA library is prepared and samples are sequenced using NGS platform. Then, quality assessment of NGS reads is carried out and reads are aligned with the reference genome. After that, variant identification and annotation is performed followed by visualization.
Next-Generation Sequencing workflow:
- Construct library
A sequencing “library” must be created from the sample. The DNA (or cDNA) sample is processed into relatively short double-stranded fragments (100–800 bp). Depending on the specific application, DNA fragmentation can be performed in a variety of ways, including physical shearing, enzyme digestion, and PCR-based ampilficati.on of specific genetic regions. The resulting DNA fragments are then ligated to technology-specific adaptor sequences, forming a fragment library. These adaptors may also have a unique molecular “barcode”, so each sample can be tagged with a unique DNA sequence.
- Clonal amplification
Prior to sequencing, the DNA library must be attached to a solid surface and clonally amplified to increase the signal that can be detected from each target during sequencing. During this process, each unique DNA molecule in the library is bound to the surface of a bead or a flow-cell and PCR amplified to create a set of identical clones. In the case of Ion Torrent technology, a process called “templating” is used to add library molecules to beads.
- Sequence library
All of the DNA in the library is sequenced at the same time using a sequencing instrument. Although each NGS technology is unique, they all utilize a version of the “sequencing by synthesis” method, reading individual bases as they grow along a polymerized strand. This is a cycle with common steps: DNA base synthesis on single stranded DNA, followed by detection of the incorporated base, and then subsequent removal of reactants to restart the cycle.
- Analyze data
Each NGS experiment generates large quantities of complex data consisting of short DNA reads. Although each technology platform has its own algorithms and data analysis tools, they share a similar analysis ‘pipeline’ and use common metrics to evaluate the quality of NGS data sets.
Advantages of NGS include:
Higher sensitivity to detect low-frequency variants1,2
Faster turnaround time for high sample volumes3
Comprehensive genomic coverage
Lower limit of detection4,5
Higher capacity with sample multiplexing
Ability to sequence hundreds to thousands of genes or gene regions simultaneously
By type of sequencing, the whole-genome sequencing segment is expected to grow substantially over the coming years. Whole-genome sequencing (WGS) has been widely accepted in providing the highest possible resolution information about COVID-19 and is believed to have the potential to transform COVID-19 infectious disease management. For instance, in early 2020, the United Kingdom government launched a new alliance “COVID-19 Genomics UK Consortium (COG-UK)” to sequence the genomes of SARS-CoV-2, the virus responsible for the current COVID-19 pandemic. The “COVID-19 Genomics UK Consortium (COG-UK)” is comprised of the NHS (National Health Service), public health agencies, the Wellcome Sanger Institute, and several academic institutions, helping in creating funds and developing diagnostics and treatment.
Moreover, numerous initiatives undertaken by universities, academic and research institutions and research establishments for utilizing WGS technology in identifying root cause of diseases are likely to propel the segment growth. For instance, in February 2021, Stanford Medicine launched an in-house service for whole-genome sequencing. Similarly, Cerba Research, announced two Covid-19 exploratory tools in January 2021, one PCR-based and the other NGS-based, to enhance research for vaccine development against infectious diseases, including Covid-19. The whole-genome sequencing of respiratory viruses and SARS-CoV-2 was greatly simplified using the NGS-based test.
NGS opens the entire spectrum of genomic alterations for the genetic analysis of complex traits and there are early publications illustrating its power. Continuing development in analytical tools will allow the promise of NGS to be realized.