Since its introduction, NGS has far exceeded the capabilities of traditional sequencing, known as Sanger sequencing. Unlike earlier methods, NGS allows for the simultaneous sequencing of millions of DNA fragments, significantly reducing time and costs while increasing the amount of data generated.
NGS technology is essential for a wide range of applications, from whole genome sequencing to more targeted studies such as exome sequencing, transcriptomics, epigenomics…
This article will guide you through the basics, current technologies, applications, and challenges of NGS, with a practical approach based on our own experience.
Main NGS Sequencing Technologies
Sequencing by Synthesis (Illumina)
Sequencing by synthesis is the most commonly used method in NGS, dominated by Illumina platforms. This technology uses a process called “sequencing by synthesis”, in which fluorescent nucleotides are incorporated into a growing DNA strand and detected using a camera that captures each step of the process.
Workflow:
First, the sample DNA is fragmented and specific adapters are added, serving three key functions:
- Attachment: Allow the DNA fragments to bind to the flow cell surface during sequencing.
- Amplification: Facilitate local amplification of each DNA fragment in the cell, creating identical fragment groups (clusters).
- Sequencing: Contain sequences that serve as binding sites for sequencing primers. Each synthesis cycle adds a nucleotide to the growing strand, emitting a fluorescent signal that is recorded.
Advantages:
High accuracy, scalability, and the ability to generate large volumes of data.
Illumina is the preferred option for whole genome sequencing projects and genetic variation studies.
Limitations:
The main limitation is the read length, which is usually short (between 50 and 300 base pairs). This can make it difficult to resolve repetitive or complex regions of the genome.
Semiconductor Sequencing (Ion Torrent)
This technology, developed by Ion Torrent, detects the release of hydrogen ions when a nucleotide is incorporated into a DNA strand. Unlike sequencing by synthesis, it does not require fluorescent nucleotides, making the process faster and less expensive.
Workflow:
Similar to Illumina, but instead of using fluorescent signals, this technology measures pH changes that occur during nucleotide incorporation.
Advantages:
Fast and low-cost, suitable for applications where time is a critical factor, such as in clinical sequencing.
Limitations:
Lower accuracy compared to Illumina, especially in detecting complex genetic variations.
Third Generation Technologies: PacBio and Oxford Nanopore
Third-generation technologies, such as Pacific Biosciences (PacBio) and Oxford Nanopore, represent a major advance in sequencing, allowing for much longer reads without the need for amplification.
PacBio:
Uses real-time sequencing (SMRT), where a DNA molecule passes through a polymerase fixed to a surface, enabling ultra-long reads (up to 20 kb or more).
Ideal for studying structural variations, repetitive regions, and for de novo whole genome sequencing.
Oxford Nanopore:
This technology enables sequencing of individual DNA molecules as they pass through a protein pore.
It is highly portable and enables real-time sequencing with variable read lengths.
- Advantages: Long reads that help resolve complex genomic regions, useful for whole genome sequencing, haplotype studies, and structural variation analysis.
- Limitations: High costs, higher error rates compared to second-generation technologies, as these are the most modern and advanced solutions.
NGS Workflow
Sample Collection and Preparation
The NGS process begins with the collection of biological samples, which may include DNA, RNA, or both, depending on the study’s goals. Samples can come from various sources, such as blood, tissue, cultured cells, or even environmental samples.
