The Guide to Understanding Target Sequencing

DNA analysis is the basis for many modern sciences, including genetics, biotechnology, medicine, and criminology. Research has made tremendous progress in genetic testing in recent years, mainly related to the elaboration of new DNA sequencing techniques.

DNA test

It all began with the publication of a preliminary, incomplete version of the human genome sequence in 2001. Since then, there has been a rapid development in genomics, including sequencing methods, which identify changes in the genes.

What exactly is target sequencing? To put it simply, it is a technique of reading sequences, particularly successive nucleotides in a DNA molecule. The most innovative method of reading sequences is Next-Generation Sequencing, which is currently conquering many laboratories across the US and the rest of the world.

The introduction of Next-Generation Sequencing methods was a significant breakthrough that revolutionized the world of molecular biology techniques and applications. Hopefully, NGS will become an indispensable tool in the diagnosis of hereditary diseases and the selection of appropriate pharmacotherapy.

What is the Next Generation Sequencing?

As one of the most modern techniques of molecular biology, NGS has a wide range of applications in various research-based and clinical settings. It is currently used for genomes and transcriptomes sequencing, research on protein-DNA / RNA interactions, checking the degree of methylation, and metagenomic studies.

The advantage of using NGS is the ability to sequence millions of DNA molecules simultaneously and affordably due to the low cost per sample analysis compared to older techniques.

Next-generation sequencing is becoming the primary tool in genetic diagnostics, replacing the existing methods whenever a disease with a genetic background is difficult to determine.  The next-generation sequencing workflow can be divided into three main steps.

The first is the isolation and construction of a DNA library, consecutively followed by the template amplification, and the last stage being the massively parallel sequencing. There are several commercially available sequencing platforms on the market today. Standard features include DNA isolation and the creation of a single-stranded DNA library.

Currently, there are three types of NGS for DNA sequencing:

  • Whole-genome sequencing (WGS), which evaluates the entire human genomic content
  • Whole-exome sequencing (WES), sequencing only the protein-coding regions of the genome.
  • Targeted sequencing, focusing on the analysis of a specific region of the genome.

What is a Genome?

The genome is the basic information about our body’s structure and functioning stored in a chemical form. The carrier of the genetic information of every living organism is deoxyribonucleic acid (DNA). Genetic information is coded in the order of the four units (nucleotides) – the letters of the genetic code – A, C, G, T appearing in the DNA chain.

DNA has a structure called a double helix that can be compared to a ladder. Each rung consists of a pair of nucleotides. There are over 3 billion such ranks in the human genome. The human genome written in text form would take 400 volumes of an encyclopedia.

Our genetic material is packed into 23 pairs of chromosomes: we get one chromosome of a given pair from each parent. The human genome consists of about 23,000 genes – regions of DNA that contain information that allows the body to produce building and regulatory proteins.

The sequence (i.e., the order of nucleotides) in DNA is transcribed into a sequence of nucleotides in the so-called messenger RNA; this, consecutively, is transcribed to the sequence of protein building units called amino acids. A genetic code is a set of rules determining how a nucleotide sequence is translated into an amino acid sequence in a protein.

What is Targeted Sequencing?

Next-Generation Sequencing, thanks to the ability to read hundreds of millions of DNA molecules simultaneously, increases throughput while dramatically reducing costs. Nevertheless, whole-genome sequencing is still quite expensive.

It does not provide the sensitivity or sufficient coverage necessary to decipher the role of single genes in complex diseases or to allow the study of rare genetic variants with low frequency. A more cost-effective way is to focus on specific regions through the use of a targeted sequencing strategy.

Targeted sequencing uses resources more efficiently, which reduces costs and simplifies further analysis, making it more manageable. Targeted sequencing utilizes several methods, such as hybridization capture and amplicon sequencing.

It involves focusing on specific regions of the genome or subset of genes, addressing desired areas of interest. The analysis may include the exome (the protein-coding part of the genome) or the custom content of specific genes.

The targeted sequencing method is most beneficial in thriving clinical and industrial settings, where time, expenses, and efficiency are highly valued and of substantial essence.


The Next-Generation Sequencing has many applications in various fields, from research to biotechnology and clinical utilization. Sequencing the genomes of pathogenic microorganisms mobilizes the search for effective prevention, diagnosis, and therapy methods.

Genome analysis allows for efficient testing of many epitopes and obtaining better vaccines. Drugs capable of selectively blocking major metabolic pathways are being designed, and molecular methods of identifying pathogens in patient samples are being introduced.

Determining the human genome sequence is the starting point for identifying mutations responsible for the development of diseases and the different susceptibility of individuals to the effects of drugs, chemical pollution of the environment, or infections.

The tests available for examining genetic predisposition, such as the occurrence of certain cancers, is continuously increasing. “Personalized” medicine is beginning to develop, in which the physician, by assessing the genetic profile of the patient, will be able to predict the likely response of the body to the therapy and select the correct type and dose of medicine. Progress is also to be expected in the field of gene therapy, drug design, and the use of human proteins for therapeutic purposes.


Giuliani M. M., Adu-Bobie J., Comanducci M., Aricò B., Savino S., Santini L., Brunelli B., Bambini S., Biolchi A., Capecchi B., et al., (2006), Proc. Natl. Acad. Sci. USA, 103, 10834-10839.

Telford J. L., (2008), Cell Host Microbe, 3, 408-416.

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