The microbiota can be defined as the community of microorganisms that live in a particular environment and as we all know, different types of microbes colonized the human body, derived from bacterial communities, microbial eukaryotes and viruses that are specific for each anatomical sites or environments. The next- generation DNA sequencing has allowed more and more advanced sampling and analysis of these complex systems by methods called culture- independent, these methods are indicating the differences in community structure between individuals, between diseased states and healthy and between anatomical sites. The advent of next generation sequencing (NGS) offered a cost-effective method that eliminated the cloning step by amplifying 16S rRNA genes using primers containing sequencing connecters and barcodes. However, this sequencing may not resolve closely related species at all times and probably miss the intra-species diversity. A shotgun sequencing was developed for direct sequencing of DNA. Metagenomics is the practice of sequencing DNA from the genomes of all organisms present in a particular sample, and has become a common method for the study of microbiota population structure and function. As sequencing-based microbiota analysis continues to be the greatest general technique across the arena, this review aim is to provide a general introduction to the technical opportunities and trials of sequence-based identification of human associated Microbiota and for understanding of the human Microbiota and their effect on human health and diseases.