**1. Introduction**

Precision medicine is a new way of practising medicine, which has been gaining strength in recent years, is based on the individual characteristics of each patient (genetic, environmental, behavioural) to optimize and customize strategies for prevention, detection and therapy [1, 2]. The molecular knowledge has contributed strongly to the advancement of precision medicine, providing specific strategies for target therapies and diagnosis of patients with cancer, Mendelian diseases and others. Statistics indicated that traditional clinical practices sometimes lead to poor health outcomes and also a waste of medical resources. It is estimated that about 75 billion US dollars per year (30% of health care expenditure) are destined for unnecessary or ineffective treatments in the USA [3].

**2. From Sanger to NGS sequencing**

know about human DNA sequence and mutations.

sequencing.

In 1908, Garrod introduced his concept 'the inborn error of metabolism' that changed the areas of biochemistry, genetics and medicine [8]. His principal contribution was the understanding about the relationship between gene-enzyme, the molecular basis of genetic diseases. Although today this concept is considered outdated because of discoveries like RNA splicing, RNAi and others, its development allowed the researchers to understand how changes in DNA sequence could cause genetic disease. This finding increased the interest of scientists to

Application of Next-Generation Sequencing in the Era of Precision Medicine

http://dx.doi.org/10.5772/intechopen.69337

295

The search to know the nucleotide sequence of DNA began in the 1960s with several studies that demonstrated new methods with different strategies [9–13], but it was in 1977 that Sanger developed the method called 'Chain-termination' that became the most used method (first generation) to sequencing DNA (**Figure 1**). The method consisted of the use of dideoxynucleotides (ddNTPs), which are deoxynucleotide analogs (dNTPs) that disrupt DNA synthesis, and the separation of the different DNA fragments in a gel. These special nucleotides were radiolabeled and therefore the sequence could be inferred after the disclosure of gel autoradiography [14]. Numerous modifications have been made in this technique to make the method more efficient, robust and sensitive. Among them are the substitution of nucleotide radiolabeled to fluorescence that allowed the sequencing reaction to occur in one tube [15], the development of the polymerase chain reaction [16], the separation of DNA fragments by capillary electrophoresis [17] and later the development of equipment that allowed the sequencing of

**Figure 1.** Timeline of DNA sequencing evolution from Sanger to NGS and the cost per raw megabase of DNA sequenced [17]. Equipment of all generations is still being improved and released commercially. Dot: milestones; rectangle: equipments; White: first-generation sequencing; Light gray: second-generation sequencing; Dark gray: third-generation

As a result of the genome project, many molecular tools have been developed and allow medical and scientific groups to improve patient management based on a better understanding of disease biology, providing a more specific and accurate prevention and treatment of diseases [4]. Precision medicine redefines the way traditional medicine is practised. There is a great deal of investment nowadays in prevention using these new technologies, as opposed to old medicine based on treatment since the disease was already evident or irreversible [2].

In recent times, Sanger sequencing, referred to as a 'first-generation' sequencing method, has partly been replaced by 'next-generation' sequencing (NGS) methods [4, 5]. NGS allows identifying biomarkers for early diagnosis as well as for personalized treatments. The emergence of NGS has changed the way clinical research, basic and applied science are done. The NGS allows producing millions of data with a smaller investment [4, 6]. Among the available NGS applications, one of them will be the resequencing of the human genome and the better genetic understanding of various human diseases. A great challenge will be the interpretation of this great number of data and its translation for the medical application [6]. One of the major near-term medical impact of the NGS revolution will be the elucidation of mechanisms of human pathogenesis, leading to improvements in the diagnosis and the selection of treatment and prevention. Thanks to second-generation sequencing technologies, it has become easier to sequence the expressed genes ('transcriptomes'), known exons ('exomes') and complete genomes of patient's samples [7].

This chapter encompasses revised concepts, applications, advances, limitations and the history of technological advances until the emergence of NGS technique in the era of precision medicine, starting with a brief history of DNA sequencing followed by a comprehensive description of most used NGS platforms, sequencing chemistries methodology and general workflows. Further topics will highlight the application of NGS towards routine practice, including variant detection, whole-genome sequencing (WGS), whole-exome sequencing (WES) and multi-gene panels. A centralized chapter describing the main NGS features in the clinic could help beginners, scientists, researchers and health care professionals, as they will be responsible for translating genomic data into genomic medicine.
