**4.1 Next-generation sequencing-based methods**

Before the advent of NGS, the Sanger sequencing method was the only protocol available to read DNA sequence or full-length 16sRNA gene amplicons. Sanger method was based on the DNA replication process and capillary electrophoresis. In this procedure, all components required for DNA synthesis, i.e., enzyme DNA polymerase, primers for 16sRNA gene, four types of deoxynucleotides (dATP, dGTP,

**13**

*Genomic Techniques Used to Investigate the Human Gut Microbiota*

con, for example, GenBank and ribosomal RNA gene bank.

dCTP, dTTP), and four types of fluorescent chain terminators (dideoxynucleotides: ddATP, ddGTP, ddCTP, ddTTP), are added to single-stranded template DNA and initiate the DNA synthesis process. Consequently, new DNA fragments of various lengths are synthesized with corresponding fluorescent chain terminators which stop further elongation of strands. Hence, randomly terminated DNA fragments are produced that are isolated with capillary gel electrophoresis. On the slab gel, four types of fluorescent dideoxynucleotides fragments can be read by a suitable laser scanning method on the basis of light emitted by them [50]. Therefore, a nucleotide sequence of 16sRNA gene amplicon can be inferred that can be searched in a large number of databases. There are many databases used for the 16sRNA gene ampli-

Sanger sequencing method not only supports the traditional metagenomic experiments but also supplemented to DGGE, TGGE, and T-RFLP methods as well as whole-genome sequencing metagenomics. The protocol includes the combination of gel and DNA sequencing based methods. In this, the isolated DNA bands from DGGE, TGGE, and T-RFLP gels are removed and sequenced by Sanger's sequencing methods. But in the case of scarcity of DNA, in a particular band, it can be further amplified by PCR and then sequenced. Sanger's sequencing method is most suitable to quantify and carry out phylogenetic identifications of the gut microbiota. Sanger method belongs to first-generation sequencing (FGS) technology, being the most important tool, and is also used for first human genome sequencing. This method is still considered as the gold standard method for long-read sequencing up to 500 nucleotides which are highly essential for genome assemblies. The main disadvantage associated with the Sanger method is its high

In the beginning of the twenty-first century, many high-throughput methods of DNA sequencing were developed, for example, pyrosequencing which is a PCR-based massively parallel sequencing platform like Roche/454 pyrosequencing exploited for investigation of gut microbiota. It provided huge genomic data related to human microbiome analyses. Pyrosequencing technique is cheap and high-throughput and requires a small amount of DNA, but short read is a major limitation of the method and unsuitable for comparisons between species within the genus and bioinformatics analysis [51]. Parallelly, other next-generation sequencing platforms for DNA sequencing are also developed such as Illumina, SOLiD, Ion Torrent, and single-molecule real-time circular consensus sequencing equipment from Pacific Biosciences and Oxford Nanopore [52]. These technologies have to make microbiome analysis very fast and easy and amass the genomic data for phylogenetic analysis. NGS has provided great speed and accuracy to cultureindependent methods used for the study of the functional diversity of microflora. Recently, MinION™ nanopore sequencing technologies used PCR-independent methods; hence, this is free from PCR-based cloning biases, such as amplification temperatures and biased primers sequences. Simultaneously, nanopore sequencing methods offer long reads, which are more suitable for genome assemblies. The above said NGS methods are applied to sequenced cloned amplicons or total community DNA [53]. These methods allow us to investigate gut microbiota qualitatively as well as quantitatively which is influenced by various perturbations, e.g.,

NGS is not only useful in phylogenetic classification but also helps in the functional analysis of microbial communities. Therefore, several supplementary technologies also emerged which can differentiate between microbial species in an ecosystem. But it requires analysis of different molecular signatures like DNA, RNAs, proteins, and metabolites generally produced by microbial communities. NGS provided the basic foundation for many omics-based methods, for example,

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

cost and time-consuming nature.

environmental factors, perturbation, and diets.

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

*Human Microbiome*

storage conditions.

bacterial species.

**4. Functional analysis of the microbial community**

**4.1 Next-generation sequencing-based methods**

*3.2.8.2 DNA microarrays*

FISH. In the studies of microbial communities, the 16S rRNA gene amplicons are prepared and denatured in a solution. After that, both fluorescent probe and DNA strands are also added in the hybridization solution. In order to allow maximum hybridization process, some cross-linking agents like aldehyde or any precipitating agent (methanol) are also added and incubated in the reaction mixture and kept at 65–75°C for 12 h [45]. After ensuring that the hybridization process is completed, the intensity of fluorescence is measured by using suitable laser available fitted in the flow cytometry instrument. The combination of FISH and flow cytometry is a sort of high-throughput method used in the genome comparison of two different species in the gut sample [46]. The FISH technique is efficiently applied to compare two types of microbial communities such as breast- and formula-fed newborns, and two different species *Bifidobacterium* and *Atopobium* are identified [47]. The merits of this method are that it is semiquantitative and rapid. Due to the availability of diverse probes for specific phyla or species, FISH can be widely used in microbiome studies. But the technique completely failed to identify de novo identification of a bacterial strain. Some researchers have used FISH to estimate the time of sample stability and change in their species compositions with the passage of time and

DNA microarray technology or DNA chip method is widely applied to learn more about the microbial ecosystem, particularly in gut microbiota. The component of the DNA microarray is a small chip containing a large number of microscopic spots on a solid surface which are used to immobilize fluorescent probes. DNA spots hold pico-level DNA, which is sufficient for hybridization process of a small part of a gene or its regulatory element with cDNA already immobilized on a DNA chip under suitable reaction environments. The microarray protocol includes the following: firstly, the 16S rRNA amplicon or extracted DNA from the samples is processed to make them fluorescent. Secondly, oligonucleotide probes are spotted and immobilized on the surface of the microarray chip [48]. Finally, hybridization is allowed between 16S rRNA amplicons and fluorescent probes. The fluorescence intensity after complete hybridization is quantified by using a laser. The microarray can identify the expression of hundreds of genes in a single experiment. The effect of *C. difficile* infection and its successful cure by fecal microbiota transplantation (FMT) is studied by microarray [49]. This method is quite fast and rapid and offers a high-throughput method for phylogenetic analysis of gut microbiota. It requires a very small amount of DNA for accurate analysis. The most noticed demerit of a microarray experiment is the possibility of cross hybridization, i.e., binding of multiple oligonucleotide probes to a single DNA fragment. In the absence of the probe, a microarray cannot identify a new

Before the advent of NGS, the Sanger sequencing method was the only protocol

available to read DNA sequence or full-length 16sRNA gene amplicons. Sanger method was based on the DNA replication process and capillary electrophoresis. In this procedure, all components required for DNA synthesis, i.e., enzyme DNA polymerase, primers for 16sRNA gene, four types of deoxynucleotides (dATP, dGTP,

**12**

dCTP, dTTP), and four types of fluorescent chain terminators (dideoxynucleotides: ddATP, ddGTP, ddCTP, ddTTP), are added to single-stranded template DNA and initiate the DNA synthesis process. Consequently, new DNA fragments of various lengths are synthesized with corresponding fluorescent chain terminators which stop further elongation of strands. Hence, randomly terminated DNA fragments are produced that are isolated with capillary gel electrophoresis. On the slab gel, four types of fluorescent dideoxynucleotides fragments can be read by a suitable laser scanning method on the basis of light emitted by them [50]. Therefore, a nucleotide sequence of 16sRNA gene amplicon can be inferred that can be searched in a large number of databases. There are many databases used for the 16sRNA gene amplicon, for example, GenBank and ribosomal RNA gene bank.

Sanger sequencing method not only supports the traditional metagenomic experiments but also supplemented to DGGE, TGGE, and T-RFLP methods as well as whole-genome sequencing metagenomics. The protocol includes the combination of gel and DNA sequencing based methods. In this, the isolated DNA bands from DGGE, TGGE, and T-RFLP gels are removed and sequenced by Sanger's sequencing methods. But in the case of scarcity of DNA, in a particular band, it can be further amplified by PCR and then sequenced. Sanger's sequencing method is most suitable to quantify and carry out phylogenetic identifications of the gut microbiota. Sanger method belongs to first-generation sequencing (FGS) technology, being the most important tool, and is also used for first human genome sequencing. This method is still considered as the gold standard method for long-read sequencing up to 500 nucleotides which are highly essential for genome assemblies. The main disadvantage associated with the Sanger method is its high cost and time-consuming nature.

In the beginning of the twenty-first century, many high-throughput methods of DNA sequencing were developed, for example, pyrosequencing which is a PCR-based massively parallel sequencing platform like Roche/454 pyrosequencing exploited for investigation of gut microbiota. It provided huge genomic data related to human microbiome analyses. Pyrosequencing technique is cheap and high-throughput and requires a small amount of DNA, but short read is a major limitation of the method and unsuitable for comparisons between species within the genus and bioinformatics analysis [51]. Parallelly, other next-generation sequencing platforms for DNA sequencing are also developed such as Illumina, SOLiD, Ion Torrent, and single-molecule real-time circular consensus sequencing equipment from Pacific Biosciences and Oxford Nanopore [52]. These technologies have to make microbiome analysis very fast and easy and amass the genomic data for phylogenetic analysis. NGS has provided great speed and accuracy to cultureindependent methods used for the study of the functional diversity of microflora. Recently, MinION™ nanopore sequencing technologies used PCR-independent methods; hence, this is free from PCR-based cloning biases, such as amplification temperatures and biased primers sequences. Simultaneously, nanopore sequencing methods offer long reads, which are more suitable for genome assemblies. The above said NGS methods are applied to sequenced cloned amplicons or total community DNA [53]. These methods allow us to investigate gut microbiota qualitatively as well as quantitatively which is influenced by various perturbations, e.g., environmental factors, perturbation, and diets.

NGS is not only useful in phylogenetic classification but also helps in the functional analysis of microbial communities. Therefore, several supplementary technologies also emerged which can differentiate between microbial species in an ecosystem. But it requires analysis of different molecular signatures like DNA, RNAs, proteins, and metabolites generally produced by microbial communities. NGS provided the basic foundation for many omics-based methods, for example, metatranscriptomics, metaproteomics, and metabolomics, which have helped us in the functional analysis of metagenome represented by a whole microbial community [54]. These methods offered a huge amount of genomic data stored in different databases that can be integrated with the help of bioinformatics tools.
