Next Generation Sequencing (NGS): origins, functioning and perspectives

From the Sanger method of the 1970s to long-read technologies and massively parallel sequencing, sequencing has evolved over time, becoming increasingly essential for medicine and research. Speed, cost reduction, and automation are the main elements that distinguish NGS,  that has multiple clinical application constantly growing

Sequencing technology encompasses a set of laboratory techniques that allow for the precise determination of the order of nucleotides in DNA or RNA. In other words, this process makes it possible to “read” the genetic code, providing crucial information about the structure and function of genes. The applications of this technology span several sectors: from medicine to genomics, evolutionary biology, and forensic science.

The evolution of sequencing: the Sanger method and automated systems

The evolution of sequencing technology has seen successive methodologies. The first effective sequencing method was developed by two-time Nobel Prize in Chemistry laureate Frederick Sanger in 1977, and is also known as “Sanger sequencing” or "dideoxy". This system, still used today in certain circumstances, is based on controlled progressive termination of DNA synthesis through three steps to determine the order of nucleotides that form a nucleic acid molecule.

In the 1980s, the Sanger method was optimized through automated systems, which, in addition to sequencers, use fluorescent dyes to label nucleotides (capillary electrophoresis systems). This made large-scale sequencing possible, increasing productivity and paving the way for large genomic projects.

Next Generation Sequencing (NGS): parallel sequencing and long-read sequencing

Starting in the 2000s, with the advent of next-generation sequencing (NGS) technologies, it became possible to sequence millions of DNA fragments in parallel, significantly reducing costs and turnaround times. This revolution, through which amplified fragments are sequenced by allowing the reading of the nucleotide sequence (A, T, C, G) of each fragment, had a huge impact in fields such as precision medicine, cancer genomics, and microbiology.

In the second decade of the millennium, a new frontier of technologies emerged, defined as third-generation, which allow long-read sequencing. Long-read sequencing makes it possible to identify and quantify isoforms on a genomic scale, overcoming the limitations of short-read technologies, which often cannot accurately reconstruct the entire structure of transcripts. These techniques thus enable reading long stretches of DNA in a single sequence, proving particularly useful for complex and repetitive genomic regions.

Contemporary sequencing technology

Since 2020, sequencing has made a further technological leap: attention has focused on improving accuracy, read speed, and cost reduction, making the technology increasingly accessible even outside large research centers. Device miniaturization and the emergence of portable platforms have paved the way for daily clinical use, allowing genetic analyses directly in the field, in clinics, or in emergency contexts.

The Next Generation Sequencing workflow

Next-generation sequencing represents a radical transformation compared to traditional methods. Its main feature is massive parallel sequencing, which allows simultaneous analysis of millions or billions of genome fragments in short times, obtaining information on the DNA of organisms, animals, and plants.

The process involves several phases

1. Library preparation, in which DNA is fragmented, tagged, and ligated to adapters
2. Clonal amplification, replicating the same sequence multiple times for better analysis;
3. Sequencing, which can occur through different techniques such as:

• ·Sequencing by synthesis (SBS) – the most common;
• Ion semiconductor sequencing;
• Nanopore sequencing;
• Real-time single-molecule sequencing (SMRT).

The clinical NGS market comprises sequencing platforms, reagents, and consumables. These tools are fundamental for genetic analysis, finding application in diagnostics, therapy, and medical research. The standard NGS workflow is structured in four phases: preparation, sequencing, data analysis, and interpretation.

Thanks to automation, artificial intelligence, and developments in bioinformatics, the entire process today is more efficient, accurate, and faster.

Applications and perspectives

The clinical applications of NGS in medicine are numerous and constantly growing. In oncology, for example, they enable identifying specific mutations in tumors, paving the way for personalized treatments (precision medicine). Next-generation sequencing is also useful for:

• Genetic testing for the identification of rare or hereditary diseases;
• Infectious disease diagnostics, thanks to its ability to identify pathogens quickly and accurately, supporting the management of epidemics and complex infections.

NGS is a pillar of precision medicine, as it allows creating therapeutic plans based on the individual genetic profile. This approach improves treatment efficacy and reduces side effects.

Technological evolution has led to device miniaturization, real-time sequencing, and integration of automated systems. All this has expanded possibilities in research, diagnostics, and drug development.

Moreover, the increasing amount of genetic data generated by next-generation sequencing has stimulated the development of increasingly advanced bioinformatic tools, indispensable for managing, interpreting, and transforming this information into rapid and targeted clinical decisions.