NGS, also known as Massively Parallel Sequencing or High-throughput Sequencing, stands for Next-Generation Sequencing. NGS is an automated, massively parallel sequencing technique that provides extremely high throughput, scalability, speed and applications. Since 2005, a number of Next Generation Sequencing (NGS) Technology and techniques have been created and put out to research any genomics question or DNA-based therapeutic activity.
NGS technologies have been successfully applied in various domains of life sciences, including; Functional genomics, transcriptomics, cancer, evolutionary biology, forensic sciences, and medicine. In a single day, researchers may sequence the whole human genome using NGS systems, which can sequence millions of DNA fragments concurrently.
History of Next-Generation Sequencing Technology
DNA sequencing techniques have only been around for around 60 years. They have advanced incredibly quickly, making them an amazing example of progress that has led to significant advancements in capability, applications, cost reduction, and high throughput. The history of DNA sequencing began with the introduction of two key techniques: Sanger sequencing and Maxam and Gilbert’s methodology.
The first human genome was sequenced in 2001 owing to advancements in polymerize chain reaction, the availability of high-quality DNA-modifying enzymes, and fluorescence automated sequencing.
The human genome was assembled using Sanger sequencing, a feat that lasted 13 years and cost around $2.7 billion. Since then, sequencing has become more faster while also becoming much cheaper. A whole human genome may now be sequenced using NGS in less than a day, and the price to sequence a full human genome fell below $1000 in 2018.
Process of Next-generation sequencing
The fundamental steps in next-generation sequencing include fragmenting DNA/RNA into separate bits, including adapters, sequencing the libraries, and putting everything back together to create a genomic sequence.
The workflow of Next-generation sequencing involves three basic steps: Library preparation, Sequencing, and Data Analysis.
From samples, such as blood, saliva, and tissue, genomic DNA is extracted. The DNA is broken up into shorter sequences, then ligated using adapters, followed by amplification and enrichment.
The platform being used affects the sequencing technique. Pyrosequencing, sequencing by synthesis, and sequencing by ligation are a few techniques. One of the most widely used technologies is sequencing by synthesis, which enables scientists to simultaneously sequence large amounts of genomic DNA at a high sensitivity to find a variety of genetic alterations, such as single-nucleotide polymorphisms (SNPs), small insertions and deletions (indels), and structural variants.
Quality checking, alignment to reference sequences, and the detection of harmful variations are all performed using bioinformatic techniques or data analysis software.
Delivery of long-read sequencing can be accomplished using two proven methods. The first method, known as nanopore sequencing, involves passing the strands through a protein nanopore and monitoring variations in electrical current as each base is taken in. The sequence is then figured out by a computer by decoding these modifications. The second type of sequencing, known as single-molecule real-time (SMRT), circularizes the strands and makes use of a polymerase to insert bases that are tagged and that, when integrated, release light. Real-time monitoring of nucleotide incorporation is accomplished by detecting the light.
New development limits are broken in next-generation sequencing technology on an almost yearly basis. The most recent advancements incorporate single-cell sequencing, which avoids ensemble average readings from a sample, which may be deceptive and provides more accurate insight into the nucleic acids of specific cells during a given phase or time point. Spatial sequencing, a new method for directly sequencing from a tissue or sample, gives researchers a spatial resolution to their data and an understanding of the makeup and interactions of the cell in its natural environment.
Platforms for Next-Generation Sequencing (NGS) Technology
The tools used for sequencing are known as NGS platforms. The technology and approach utilized for bulk sequencing vary amongst the many platforms that are currently on the market. These are the primary NGS platforms:
Illumina, Inc is the leading manufacturer of different sequencing tools. It was founded in 1998 in San Diego, California. It can execute extensive sequencing with high-quality reads at a more reasonable cost.
Illumina sequencing process is based on Sequence by Synthesis (SBS) principle. Incorporating nucleotides into a nucleic acid chain while simultaneously identifying them is the procedure.
DNA polymerase, primers, and 4 dNTP with base-specific fluorescent markers make up the reaction system.
Chemically altered nucleotides attach to the DNA template strand through complementarity, in essence. These nucleotides include both a fluorescent tag and a reversible terminator that prevents the inclusion of the next base.
The fluorescent signal shows which nucleotide has been added, and the terminator is broken to allow the attachment of the following base and completion of the sequencing.
The most recent sequencing technologies are able to read both ends of a fragment and create DNA sequences in a paired-end method. The improper cleavage of the fluorescent label or termination moieties led to signal degradation and dephasing. The sequencing platforms’ typical error rates range from 1 to 1.5%.
In 2011, Ion Torrent made its debut. Ion Torrent is an SBS-based method that generates nucleotide sequences using pH readings.
The production of a hydrogen ion during the integration of a dNTP allows for the detection of nucleotide addition. A strong positive voltage is created throughout the process due to the release of H+ ions, which alters the pH of the surrounding area and is picked up by a device.
Ion Torrent produces sequencing reads of various lengths. Ion Torrent sequencing devices are unable to produce sequencing from a fragment’s ends.
The first commercially available 454 Sequencer was the Roche GS-FLX 454 Genome Sequencer, which was introduced in 2004. James D. Watson’s genome was the second to be sequenced entirely using this technique.
The Pyrosequencing technique, which is used by this platform, is based on the detection of pyrophosphate generated after the integration of a nucleotide in the freshly manufactured DNA strand.
The Platform can produce around 1 million reads per run and has a maximum read length of 1000 base pairs for amplicons and 600 base pairs for genomic DNA. The average read length and accuracy increased to 700 bp and 99.997%, respectively, with Roche’s 2008 introduction of the improved 454 GS FLX Titanium technology.
The Stanford Genome Technology Center is home to the 2010-founded GenapSys. The thermal detection of nucleotide incorporations was embedded into the GenapSys Sequencer to improve the SBS method.
It is lightweight (less than ten pounds), inexpensive, and simple to operate, making it suitable even for those new to the field of genomics. The electrical chip has millions of sensors, each with a single bead covered in clonal copies of a nucleotide sequence in the thousands. The complementary DNA strand expands when the DNA bases are poured across the chip in a certain order, and variations in resistance indicate successful inclusion. The chip is offered in three different configurations, each with a different number of sensors: 1 million, 16 million, and 144 million.
Pacific Biosciences and the Oxford Nanopore: Single-Molecule Sequencing
These more recent platforms are capable of resolving issues with older sequencing techniques, including as reading mistakes in short reads or even the need for bench labor for sample preparation.
The Single-Molecule Sequencing (3rdGS) method is also known as the single template method. The basic principle is to measure changes in the electrical properties of DNA as it translocate through channels.
The most recent PacBio sequencer (Sequel IIe System, launched on October 5, 2020) can create 4 million readings with more than 99% accuracy in under 30 hours, as an illustration of how SMRT can produce tens of kilobases long reads. Comparing this technology to Nanopore and Illumina, it was shown to have greater contiguity (N50), accuracy (quality score), and completeness (genome size).
Application of Next Generation Sequencing
- Whole genome sequencing to determine an organism’s complete DNA sequence
- Whole exome sequencing to focus on the coding regions of the genome
- Targeted sequencing to study specific genomic regions of interest
- Epigenomics to evaluate epigenetic modifications
- RNA Sequencing for transcriptome profiling of coding and noncoding regions, identifying genes in specific cell types and determining genetic alterations like gene fusions and single nucleotide variants (SNVs)
- To help unlock the potential of every sample, Roche offers clinicians and researchers an extensive portfolio of products for their NGS needs.