**Abstract**

The human gut is the complex microbial ecosystem comprises more than 100 trillion microbes also known as microbiota. The gut microbiota does not only include about 400–500 types of bacterial strains, but it also contains archaea, bacteriophage, fungi, and protozoa species. In order to complete the characterization of the gut microbial community, we need the help of many culture-dependent and cultureindependent genomic technologies. Recently, next-generation sequencing (NGS), mediated metagenomics that rely on 16S rRNA gene amplification, and wholegenome sequencing (WGS) have provided us deep knowledge related to important interactions such as host-microbiota and microbe-microbe interactions under various perturbation inside the gut. But, we still lack complete knowledge related to unique gene products encoded by gut meta-genome. Hence, it required the application of high-throughput "omics-based" methods to support metagenomics. Currently, a combination of high-throughput culturing and microfluidics assays is providing a new method to characterize non-amenable bacterial strains from the gut environment. The recent additions of artificial intelligence and deep learning to the area of microbiome studies have enhanced the capability of identification of thousand microbes simultaneously. Given above, it is necessary to apply new genome editing tools that can be used to design the personalized microflora which can be used to cure lifestyle-related diseases.

**Keywords:** culturomics, gut microbiota, human microbiome, metagenomics, metaproteomics, metabolomics, microfluidics, "multi-omics", personalized diet

## **1. Introduction**

In the beginning of the twenty-first century, the human genome was sequenced. The main aim of this gigantic scientific effort was to identify all genes present in the human genome, also considered as the "blueprint of human life." Since then, most of the efforts are focused on the identification of all genes and annotate their functions which are responsible for genetic variation prevailed in human physiology and its association with diseases [1]. Currently, many experiments have proved that the gut microbes are more responsible than host genetics in the development of life stylerelated diseases. Hence, it becomes essential to investigate the crucial roles played by gut microbes in health and diseases. The human gut is a complex microbial ecosystem which is comprised of approximately 100 trillion microbes collectively known as "gut microbiota" [1]. It does not only include about 400–500 types of bacterial species but

also contains archaea, bacteriophage, fungi, and protozoa species [2]. According to a rough estimation, the human gut microbiome contains almost 3.3 million genes which are 150 times more than total human genes present in the human genome. Currently, gut microflora is also considered as "gold mines" because of its commercial value in the area of biopharmaceuticals and bioactive products. In order to complete the characterization of the gut microbial community and its mysteries, we need the help of many traditional and modern genomic technologies developed in due course of time. However, the study of the human microbiome is relatively a newly emerging area in the area of human biology, thus called the "forgotten organ" in the human body.

The study of the human microbiome was started with the help of reductionist approaches such as identification and characterization of a single bacterial strain by using culture media and microscopes. Initially, only culturable bacteria could only be identified and phylogenetically classified. It is well known that more than 40% of gut microbes cannot grow outside the natural environment. Hence, both culture-dependent and culture-independent analytical methods are applied that have improved our knowledge related to human gut microbiota. Recently, nextgeneration sequencing (NGS) has revolutionized all areas of biological sciences including the human gut microbiome. This also supports the most traditional metagenomic technique based on 16sRNA gene amplification via polymerase chain reaction (PCR) and whole-genome sequencing (WGS) also. However, both culturedependent and culture-independent techniques have provided the snapshot of the gut microbial community, but they are still hazy in respect of host-microbiota and microbe-microbe interactions that make stable conditions of gut microbial communities under the influence of various perturbations such as environmental factors, diets, and drugs. In the last 20 years, it becomes apparent that gut microbes add in the metabolism and contribute to strengthening the host's immune system. The human gut microbiota constitutes a metagenome that encodes an intricate network of genes, proteins, and metabolites. In order to functionally characterize human microbiome, it requires applications of many supplementary highthroughput "omics-based" methods, e.g., metaproteomics, metatranscriptomics, and metabolomics.

Recently, several labs the world over have adopted new emerging technologies to support metagenomics consequently; it amasses the terabits data in various genomic databases. To retrieve meaningful information from a large amount of multi-omics data, the application of a high level of computational and bioinformatics knowledge is required. In view of the recent explosion of data in every field, machine learning and deep learning come forward for the rescue of scientists. Therefore, different algorithms have been created, tested, and applied to huge microbiome data to identify the results of numerous microbial strains. But the next aim of all plethora of technologies is to unravel the significant contribution of gut microbiota to human biochemistry and physiology, and ultimately, this knowledge can be translated to improve human health and reduce lifestyle-related pandemic prevailed worldwide. In view of the above facts, the current chapter describes a set of analytical methods that are used to dig deep into the human gut microbial community. These methods are exploited in phylogenetic classification and functional characterization of gut microbiota.

#### **2. A brief history of the human microbiome study**

The field of the human microbiome is closely associated with microbiology; hence, its study was started in the seventeenth century. Antonie van Leeuwenhoek, who is also considered the father of microbiology, discovered oral microbes by using a simple microscope and called them "animalcules" in 1676. In the 1800s, Robert Koch developed the investigation technique for anthrax. The pioneering

**5**

*Genomic Techniques Used to Investigate the Human Gut Microbiota*

work of Pasteur, Koch, Escherich, and Kendall founded a strong base of microbiome research; hence, they are able to identify and count a large number of bacterial strains. In 1907, Metchnikoff proposed that lactic bacteria can ward off against harmful or putrefying bacteria from the gut [2]. Joshua Lederberg for the first time used the term "microbiome" for gut microbial community, and its relationship with

In the beginning, only culturable bacterial species were studied, but there are a large number of microbes that are not grown inside the lab environment. That was revealed when the number of microorganisms observed by the microscope did not match with a number of microorganisms that grow on the media plate [4]. In 1970, Carl Woese suggested that ribosomal RNA genes can be used as molecular markers for bacterial classification [5]. Thus, scientists have developed the culture-independent technique based on amplification of 16S rRNA gene by PCR method and its sequencing by Sanger method. These strategies are used to classify gut microorganisms phylogenetically and then annotate their functions in a particular natural microbial ecosystem [6]. It has revolutionized the field of "microbiome research." Other culture-independent techniques, which significantly influence the taxonomic research, were the PCR, rRNA gene cloning and sequencing, fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE), restriction fragment length polymorphism (RFLP), and terminal restriction fragment length polymorphism (T-RFLP). But these techniques could not reveal the metabolic and ecological functions of microorganisms. In order to ascertain the function of individual bacterial strain in the gut ecosystem, germ-free mouse

But due to cumbersome and time-consuming methods of traditional metagenomic techniques, new methods based on NGS have taken over the central stage to investigate the microbial communities [7]. Currently, sequencing-based techniques are used to classify numerous uncultivable microbes. Most recently, mass spectroscopy (MS) and one of its variants, i.e., matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF)-based "omics"-based high-throughput methods, have been applied to functional characterization of microbial communities [8]. These sophisticated technologies have amassed a huge amount of genomic data that needs to be annotated by computer-based systems biology approaches. The systems biology will provide a holistic picture of the microbial community inside the human gut. Currently, seven major groups such as *Firmicutes*, *Bacteroidetes*, *Proteobacteria*, *Fusobacteria*, *Verrucomicrobia*, *Cyanobacteria*, and *Actinobacteria* which constitute a major chunk of gut microbes have been recognized, but out of these two phyla, namely, *Firmicutes* and *Bacteroidetes*, include

Initially, culture and biochemical typing were the standard methods to identify any new bacterial species. To know more about the human gut microbes' diversity, its compositions, and relationships with various diseases, thus, many other techniques are also developed. The evolution of various methods applied to investigate the human gut microbiota is described above. Recently, significant advancements have been made in the area of sequencing-based genome technologies including metatranscriptomics, proteomics, and metagenomics, which are further supported by culturomics and computational biology for studies of human gut microbiome research. These techniques are rapid and, hence, provided a huge wealth of genomic

the host. The microbial community can be defined as "the set of organisms (in this case, microorganisms) coexisting in the same space and time" [3].

*DOI: http://dx.doi.org/10.5772/intechopen.91808*

models were also developed.

most of the gut bacteria species [9].

**3. Methodology for human gut microbiome studies**

#### *Genomic Techniques Used to Investigate the Human Gut Microbiota DOI: http://dx.doi.org/10.5772/intechopen.91808*

*Human Microbiome*

and metabolomics.

also contains archaea, bacteriophage, fungi, and protozoa species [2]. According to a rough estimation, the human gut microbiome contains almost 3.3 million genes which are 150 times more than total human genes present in the human genome. Currently, gut microflora is also considered as "gold mines" because of its commercial value in the area of biopharmaceuticals and bioactive products. In order to complete the characterization of the gut microbial community and its mysteries, we need the help of many traditional and modern genomic technologies developed in due course of time. However, the study of the human microbiome is relatively a newly emerging area in the area of human biology, thus called the "forgotten organ" in the human body. The study of the human microbiome was started with the help of reductionist approaches such as identification and characterization of a single bacterial strain by using culture media and microscopes. Initially, only culturable bacteria could only be identified and phylogenetically classified. It is well known that more than 40% of gut microbes cannot grow outside the natural environment. Hence, both culture-dependent and culture-independent analytical methods are applied that have improved our knowledge related to human gut microbiota. Recently, nextgeneration sequencing (NGS) has revolutionized all areas of biological sciences including the human gut microbiome. This also supports the most traditional metagenomic technique based on 16sRNA gene amplification via polymerase chain reaction (PCR) and whole-genome sequencing (WGS) also. However, both culturedependent and culture-independent techniques have provided the snapshot of the gut microbial community, but they are still hazy in respect of host-microbiota and microbe-microbe interactions that make stable conditions of gut microbial communities under the influence of various perturbations such as environmental factors, diets, and drugs. In the last 20 years, it becomes apparent that gut microbes add in the metabolism and contribute to strengthening the host's immune system. The human gut microbiota constitutes a metagenome that encodes an intricate network of genes, proteins, and metabolites. In order to functionally characterize human microbiome, it requires applications of many supplementary highthroughput "omics-based" methods, e.g., metaproteomics, metatranscriptomics,

Recently, several labs the world over have adopted new emerging technologies to support metagenomics consequently; it amasses the terabits data in various genomic databases. To retrieve meaningful information from a large amount of multi-omics data, the application of a high level of computational and bioinformatics knowledge is required. In view of the recent explosion of data in every field, machine learning and deep learning come forward for the rescue of scientists. Therefore, different algorithms have been created, tested, and applied to huge microbiome data to identify the results of numerous microbial strains. But the next aim of all plethora of technologies is to unravel the significant contribution of gut microbiota to human biochemistry and physiology, and ultimately, this knowledge can be translated to improve human health and reduce lifestyle-related pandemic prevailed worldwide. In view of the above facts, the current chapter describes a set of analytical methods that are used to dig deep into the human gut microbial community. These methods are exploited in phylogenetic classification and functional characterization of gut microbiota.

The field of the human microbiome is closely associated with microbiology; hence, its study was started in the seventeenth century. Antonie van Leeuwenhoek, who is also considered the father of microbiology, discovered oral microbes by using a simple microscope and called them "animalcules" in 1676. In the 1800s, Robert Koch developed the investigation technique for anthrax. The pioneering

**2. A brief history of the human microbiome study**

**4**

work of Pasteur, Koch, Escherich, and Kendall founded a strong base of microbiome research; hence, they are able to identify and count a large number of bacterial strains. In 1907, Metchnikoff proposed that lactic bacteria can ward off against harmful or putrefying bacteria from the gut [2]. Joshua Lederberg for the first time used the term "microbiome" for gut microbial community, and its relationship with the host. The microbial community can be defined as "the set of organisms (in this case, microorganisms) coexisting in the same space and time" [3].

In the beginning, only culturable bacterial species were studied, but there are a large number of microbes that are not grown inside the lab environment. That was revealed when the number of microorganisms observed by the microscope did not match with a number of microorganisms that grow on the media plate [4]. In 1970, Carl Woese suggested that ribosomal RNA genes can be used as molecular markers for bacterial classification [5]. Thus, scientists have developed the culture-independent technique based on amplification of 16S rRNA gene by PCR method and its sequencing by Sanger method. These strategies are used to classify gut microorganisms phylogenetically and then annotate their functions in a particular natural microbial ecosystem [6]. It has revolutionized the field of "microbiome research." Other culture-independent techniques, which significantly influence the taxonomic research, were the PCR, rRNA gene cloning and sequencing, fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE), restriction fragment length polymorphism (RFLP), and terminal restriction fragment length polymorphism (T-RFLP). But these techniques could not reveal the metabolic and ecological functions of microorganisms. In order to ascertain the function of individual bacterial strain in the gut ecosystem, germ-free mouse models were also developed.

But due to cumbersome and time-consuming methods of traditional metagenomic techniques, new methods based on NGS have taken over the central stage to investigate the microbial communities [7]. Currently, sequencing-based techniques are used to classify numerous uncultivable microbes. Most recently, mass spectroscopy (MS) and one of its variants, i.e., matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF)-based "omics"-based high-throughput methods, have been applied to functional characterization of microbial communities [8]. These sophisticated technologies have amassed a huge amount of genomic data that needs to be annotated by computer-based systems biology approaches. The systems biology will provide a holistic picture of the microbial community inside the human gut. Currently, seven major groups such as *Firmicutes*, *Bacteroidetes*, *Proteobacteria*, *Fusobacteria*, *Verrucomicrobia*, *Cyanobacteria*, and *Actinobacteria* which constitute a major chunk of gut microbes have been recognized, but out of these two phyla, namely, *Firmicutes* and *Bacteroidetes*, include most of the gut bacteria species [9].

#### **3. Methodology for human gut microbiome studies**

Initially, culture and biochemical typing were the standard methods to identify any new bacterial species. To know more about the human gut microbes' diversity, its compositions, and relationships with various diseases, thus, many other techniques are also developed. The evolution of various methods applied to investigate the human gut microbiota is described above. Recently, significant advancements have been made in the area of sequencing-based genome technologies including metatranscriptomics, proteomics, and metagenomics, which are further supported by culturomics and computational biology for studies of human gut microbiome research. These techniques are rapid and, hence, provided a huge wealth of genomic data related to uncultured microorganisms. This helped us in the identification of new microbe species inside the gastrointestinal tract. But there are many important issues associated with the accurate and proper investigation of a gut ecosystem like sample preparation, storage, and handling from the human as well as animal subjects. In the current chapter, total techniques under three major headings (1) culture-dependent methods, (2) culture-independent genomic technologies, and (3) latest techniques are described (**Figure 1**).
