**Abstract**

Metagenomic approaches are a growing branch of science and have many applications in different fields. Metagenomics seems to be the ideal cultureindependent technique for unraveling the biodiversity of soils and to study how this biodiversity is affected with continuously changing conditions. In addition, its application in clinical and diagnostic approaches was reported. The emergence of several next-generation sequencing (NGS) strategies enriched the metagenomics. The combination between NGS and metagenomic approaches helped the investigators resolve several issues regarding the microbial diversity and the functions and relationships among different microbial flora. A number of NGS approaches were developed including Roche/454 pyrosequencing, Illumina/Solexa sequencing, and Applied Biosystems/SOLiD sequencing. In this chapter, different NGS platforms are discussed in terms of principle, advantages, and limitations. In addition, thirdgeneration sequencing technologies are also addressed.

**Keywords:** high throughput, metagenomics workflow, sequencing approaches, metagenomic data analysis

### **1. Introduction**

The development of next-generation sequencing (NGS) techniques provides highthroughput sequence analysis with the ability to simultaneously and independently sequence billions of DNA molecules. The combination between such technologies and metagenomic approaches helped the investigators study the microbial diversity and understand the functions and relationships among different microbial flora [1]. The use of metagenomic NGS by microbiologists overcomes several limitations and secured the unbiased methods to study the microbial flora in any given environment [2]. Thus, the dynamic of complex communities particularly those with non-cultivable microorganisms can be resolved [3, 4]. In addition, metagenomic NGS found its way in the field of clinical and diagnostic approaches [5, 6]. In the clinical field, NGS was used to inform the real-time incidence and prevention response to human parainfluenza 3 virus infections [7] and for cerebrospinal fluid diagnostics [8].

Several NGS platforms were developed since 2006 with numerous applications in genetic and biological research fields. Of these platforms, the most commonly used include Roche/454 pyrosequencing, Illumina/Solexa sequencing, and Applied Biosystems/SOLiD sequencing. The principle of all these NGS depends on the detection of luminescent signals released by the base incorporation during the

sequencing process [4]. They also share the same workflow which in the order, DNA extraction, library construction, DNA template preparation, and automated sequence analysis [9]. In this chapter, different NGS platforms are discussed in terms of principle, advantages, and limitations. In addition, third-generation sequencing technologies are also addressed.
