*3.2.1 Sample collection and standardization methods*

The sample preparation is a very crucial and important step of any microbial or biochemical analysis that determines the accuracy and efficacy of any simple or sophisticated analytical technique. In the human microbiome studies, there are two major types of samples, namely, stool and mucosal biopsy. However, the mucosal biopsy sample must be preferred, but their availability and handling are not easy. Ideally, stool samples must be used in conjunction with the mucosal samples [21, 22]. Several proofs of investigation have shown that there are great ambiguities prevailed between the presence of microbiota in mucosal and stool samples. Sample collection and their storage conditions also influence the final results in terms of the genetic composition of gut microbes. It has been noticed that the populations of the two most abundant gut microbial species such as *Firmicutes* to *Bacteroidetes* are affected with storage temperature in the fecal sample [23]. The sample processing methods are also held responsible for the variations in results. Hence, different consortiums associated with large-scale investigation of the gut microbiome have suggested that we must adopt the standard and calibrated protocols for sample processing [24]. Therefore, many kits are developed, for example, Qiagen QIAamp DNA Stool Mini Kit (QIAG) has significantly improved the DNA extraction and reproducibility of results from fecal samples. Moreover, researchers have also recommended other methods, namely, phenol/chloroform (PHEC), chaotropic (CHAO), and THSTI. Their comparative efficacies and performance were analyzed in terms of the final yield of DNA [26]. Currently, one more DNA/ RNA Extraction Kit (TS), i.e., TianLong Stool, is also used, which mainly acts on mechanical shearing and bead beating method. It also provided good reproducible results. However, comparative studies reflect that the TS kit offers a higher quantity of nucleic acids than the other extraction kits. Conclusively, we can say that, standard protocols that are available in the form of kits that save our time and efforts of researchers [25].

## *3.2.2 Metagenomic analysis of microbial community*

In order to overcome the drawbacks of traditional culture-based protocols, microbiologists have developed several advanced culture-independent methods to know the composition of gut microbiota. In this series, metagenomics was the first technique by which 80% of uncultured microbes are phylogenetically identified.

**9**

to template DNA strands [31].

*3.2.3 Real-time PCR*

*Genomic Techniques Used to Investigate the Human Gut Microbiota*

area of human microflora investigations in the last two decades.

This culture-independent technique for microbial growth has revolutionized the

The classical techniques of metagenomics rely on the 16S ribosomal RNA (16S rRNA) gene. The 70S ribosome is the major component of prokaryotic cells and involved in protein synthesis which is highly conserved processes in all bacterial cells. The major function of 16S rRNA is the regulation of protein synthesis. During protein synthesis process, 3′ end of 16S RNA combines with the ribosomal proteins S1 and S21 involved in activation and initiation of protein synthesis. Although 16S rRNA is highly conserved among microbial species, it also contains few hypervariable regions that offer phylogenetic linkage; hence, it is also proven to be helpful in the classification of enormous microbial diversities that prevailed on earth [26]. With the development of DNA sequencing methods, 16S rRNA gene amplicons are isolated and sequenced; hence, it now becomes the most successful and prevalent culture-independent method for taxonomic classification of microorganisms. After the availability of PCR-based cloning and 16S rRNAs, gene sequencing has revolutionized the area of taxonomic classification of uncultured bacterial strains in the last two decades [27]. The metagenomic protocols include the extraction of nucleic acid from the sample followed by PCR amplification of species-specific 1500-bp-long whole 16S ribosomal RNA genes [28]. It also contains highly hypervariable regions (the V4–V5 region out of nine short hypervariable regions from V1 to V9). PCR-based amplification is carried out by using universal and specific primers, and after that, physical

separation of DNA fragments are carried out on electrophoresis gels [29].

Initially, 16S ribosomal RNA gene amplification was based on cloning in a suitable host, e.g., *Escherichia coli*, and then sequencing by Sanger sequencing method. After availability of PCR based cloning of 16S ribosomal RNA gene and then, sequencing of clones (amplicons) by using any DNA sequencing method. These methods have tremendously enhanced phylogenetically the identification of the gut microbiota [30]. At that time, the pace and cost of sequencing were the great impediments that could be overcome by the advent of NGS. It is now well known that PCR-mediated protocols used for characterization of microbial diversity have certain demerits. These are attributed to PCR-based amplification of 16S rRNA gene, which is a multi-step process that introduced several ambiguities into the final results, and it became more error prone due to the PCR-based sequencing method, e.g., pyrosequencing [33]. Generally gene-specific amplifications are primer based which must be appropriate for all major taxa. Furthermore, the amplified DNA fragments can harbor mutations because of the nonspecific binding of PCR primers

Recently, next-generation DNA sequencing has made metagenomic and wholegenome sequencing metagenomic methods more rapid and highly sophisticated. The latest sequencing methods such as 454 pyrosequencing, Illumina, SOLiD, Ion Torrent, and single-molecule real-time (SMRT) circular consensus sequencing equipment from Pacific Biosciences [32] and Oxford Nanopore have provided more pace and deep analytic power to the analyzed gut microbiome [33]. More recently, the application of Oxford Nanopore in gut microbe analysis can overcome the abovementioned PCR-based limitations such as PCR temperature, cloning, and long and deep sequencing by MinION™ nanopore sequencing technologies.

It is well known that PCR is a nonquantitative technique, but its variant, realtime PCR also known as quantitative PCR (qPCR), is used for microbiome analysis particularly for phylogenetic analysis. It can be used quantitatively and semiquantitatively depending upon the applications; qPCR can quantify the amount of DNA

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

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

*Human Microbiome*

microfluidics-based model (human-microbial cross talk (HuMiX)). The HuMiX provide gastrointestinal-like environment for the co-growth of human epithelial cell and obligate anaerobe *Bacteroides caccae* cells [18]. Recently developed iChip containing multiple microchambers which are further divided into hundreds of miniature multiple cells has been used to grow bacteria. This technique mainly acts by providing a selective supply of nutrients to an inoculated single bacterial cell onchip. Another chip-based method l-tip also acts on the same principles as iChip, but it allows bacterial cell multiplication in a gel and supplies required nutrients which are essential for growth [19]. Microfluidics is the combination of gel-based methods and sophisticated instruments, for example, first we grow a single bacterial cell, then amplify its genome, and, finally, sequence its genome that helps in identifying new species [20]. Recently, TM7, bacterium, and *Sulcia muelleri* could be identified which produced very unique metabolites. By using the same method, 34 various

The sample preparation is a very crucial and important step of any microbial or biochemical analysis that determines the accuracy and efficacy of any simple or sophisticated analytical technique. In the human microbiome studies, there are two major types of samples, namely, stool and mucosal biopsy. However, the mucosal biopsy sample must be preferred, but their availability and handling are not easy. Ideally, stool samples must be used in conjunction with the mucosal samples [21, 22]. Several proofs of investigation have shown that there are great ambiguities prevailed between the presence of microbiota in mucosal and stool samples. Sample collection and their storage conditions also influence the final results in terms of the genetic composition of gut microbes. It has been noticed that the populations of the two most abundant gut microbial species such as *Firmicutes* to *Bacteroidetes* are affected with storage temperature in the fecal sample [23]. The sample processing methods are also held responsible for the variations in results. Hence, different consortiums associated with large-scale investigation of the gut microbiome have suggested that we must adopt the standard and calibrated protocols for sample processing [24]. Therefore, many kits are developed, for example, Qiagen QIAamp DNA Stool Mini Kit (QIAG) has significantly improved the DNA extraction and reproducibility of results from fecal samples. Moreover, researchers have also recommended other methods, namely, phenol/chloroform (PHEC), chaotropic (CHAO), and THSTI. Their comparative efficacies and performance were analyzed in terms of the final yield of DNA [26]. Currently, one more DNA/ RNA Extraction Kit (TS), i.e., TianLong Stool, is also used, which mainly acts on mechanical shearing and bead beating method. It also provided good reproducible results. However, comparative studies reflect that the TS kit offers a higher quantity of nucleic acids than the other extraction kits. Conclusively, we can say that, standard protocols that are available in the form of kits that save our time and

bacterial strains are identified and phylogenetically classified.

*3.2.1 Sample collection and standardization methods*

**3.2 Culture-independent methods**

**8**

efforts of researchers [25].

*3.2.2 Metagenomic analysis of microbial community*

In order to overcome the drawbacks of traditional culture-based protocols, microbiologists have developed several advanced culture-independent methods to know the composition of gut microbiota. In this series, metagenomics was the first technique by which 80% of uncultured microbes are phylogenetically identified.

This culture-independent technique for microbial growth has revolutionized the area of human microflora investigations in the last two decades.

The classical techniques of metagenomics rely on the 16S ribosomal RNA (16S rRNA) gene. The 70S ribosome is the major component of prokaryotic cells and involved in protein synthesis which is highly conserved processes in all bacterial cells. The major function of 16S rRNA is the regulation of protein synthesis. During protein synthesis process, 3′ end of 16S RNA combines with the ribosomal proteins S1 and S21 involved in activation and initiation of protein synthesis. Although 16S rRNA is highly conserved among microbial species, it also contains few hypervariable regions that offer phylogenetic linkage; hence, it is also proven to be helpful in the classification of enormous microbial diversities that prevailed on earth [26]. With the development of DNA sequencing methods, 16S rRNA gene amplicons are isolated and sequenced; hence, it now becomes the most successful and prevalent culture-independent method for taxonomic classification of microorganisms. After the availability of PCR-based cloning and 16S rRNAs, gene sequencing has revolutionized the area of taxonomic classification of uncultured bacterial strains in the last two decades [27].

The metagenomic protocols include the extraction of nucleic acid from the sample followed by PCR amplification of species-specific 1500-bp-long whole 16S ribosomal RNA genes [28]. It also contains highly hypervariable regions (the V4–V5 region out of nine short hypervariable regions from V1 to V9). PCR-based amplification is carried out by using universal and specific primers, and after that, physical separation of DNA fragments are carried out on electrophoresis gels [29].

Initially, 16S ribosomal RNA gene amplification was based on cloning in a suitable host, e.g., *Escherichia coli*, and then sequencing by Sanger sequencing method. After availability of PCR based cloning of 16S ribosomal RNA gene and then, sequencing of clones (amplicons) by using any DNA sequencing method. These methods have tremendously enhanced phylogenetically the identification of the gut microbiota [30]. At that time, the pace and cost of sequencing were the great impediments that could be overcome by the advent of NGS. It is now well known that PCR-mediated protocols used for characterization of microbial diversity have certain demerits. These are attributed to PCR-based amplification of 16S rRNA gene, which is a multi-step process that introduced several ambiguities into the final results, and it became more error prone due to the PCR-based sequencing method, e.g., pyrosequencing [33]. Generally gene-specific amplifications are primer based which must be appropriate for all major taxa. Furthermore, the amplified DNA fragments can harbor mutations because of the nonspecific binding of PCR primers to template DNA strands [31].

Recently, next-generation DNA sequencing has made metagenomic and wholegenome sequencing metagenomic methods more rapid and highly sophisticated. The latest sequencing methods such as 454 pyrosequencing, Illumina, SOLiD, Ion Torrent, and single-molecule real-time (SMRT) circular consensus sequencing equipment from Pacific Biosciences [32] and Oxford Nanopore have provided more pace and deep analytic power to the analyzed gut microbiome [33]. More recently, the application of Oxford Nanopore in gut microbe analysis can overcome the abovementioned PCR-based limitations such as PCR temperature, cloning, and long and deep sequencing by MinION™ nanopore sequencing technologies.

#### *3.2.3 Real-time PCR*

It is well known that PCR is a nonquantitative technique, but its variant, realtime PCR also known as quantitative PCR (qPCR), is used for microbiome analysis particularly for phylogenetic analysis. It can be used quantitatively and semiquantitatively depending upon the applications; qPCR can quantify the amount of DNA in the stool or gut mucosa samples. In this technique, fluorescent probes or dye molecules are used that intercalate between the double strand of DNA molecules or 16 s RNA amplicons. These probes send a strong signal, and its intensity is directly proportional to the amount of DNA sample present. Sometimes sequence-specific oligonucleotide probes are linked with molecular markers or complementary DNA sequence [34]. The primers designing is a crucial step in the RT-PCR technique; therefore, primers must be specific for all bacterial phyla or taxa or species present in a sample [35]. Real-time PCR has been used to investigate the state of the ecological environment in normal and obese persons [36]. Quantitative PCR technique is also used solely or in combination with other gel and non-gel-based techniques. This combination of protocols is used to understand the functional microbial diversity of gut microbiota in the patient of age and effect of antibiotics on gut microbes [37], for example, DGGE and qPCR.

Real-time PCR-based methods are suitable for the prediction of accurate phylogenetic analysis. The appropriate primers provide great help to know the composition of a microbial community and microbial load. The protocol is simple to complex, and all chemicals and consumables are easily available in laboratories. But, this is also suffering due to PCR biases, which percolate at each step of the protocol. Quantitative PCR cannot be used to detect new bacterial strains in the gut microbiota without prior information of primers or probe.
