The microbiome has been called another organ or sometimes referred to as our second genome, the genes of microbes that make up the microbiota outnumber human genes by more than 100-fold, with over 3 million bacterial genes in the gut alone. Over 99% of the bacteria in the gut are anaerobes, many of which have not been studied thoroughly because they cannot be cultured outside of their hosts. As traditional isolation and culture methods limiting the study of gut microbiota, microbiome analysis that recover the full-scale genetic information of gut microbiota has offer convenient, efficient and comprehensive solutions, which benefit greatly from methods including 16S/18S/ITS amplicon sequencing and shotgun metagenomic sequencing that based on next-generation sequencing (NGS) technology, are throwing light upon the research into gut microbiota, to offer insight into human gut microbial complexity among different hosts with hoping that such knowledge could address the health issues that people suffer from.
16S/18S/ITS Amplicon Sequencing has now been a well-established method for microbial identification and quantitation, taxonomy and phylogeny studies of gut microbiota - 16S rRNA sequencing aimed at archaea and bacteria, while 18S rRNA sequencing is commonly used for eukaryotes and ITS sequencing for fungi mostly. These genes are composed of conserved regions and highly variable regions, which is the foundation making it a practical method for the exploration of gut microbial diversity.
Whole-genome sequencing delivers a comprehensive view of the entire genome, which works as a powerful tool in multiple areas including diagnosis, tracking disease outbreaks, characterizing causative mutations and identifying drug-resistance genes.
Instead of merely revealing phylogenetic information like the precursor technologies such as amplicon sequencing (e.g. 16S rRNA sequencing), which can also be difficult when unknown microbes are being studied for lack of informative markers or primers, shotgun metagenomic sequencing (also known as whole metagenome shotgun or WMGS sequencing), on the other hand, can give a broader view of information and indications of gene functions - Shotgun metagenomic sequencing genetically analyzes DNA from different types of microbes in the gut community to understand what organisms are present and their physiology. For instance, the use of shotgun metagenomics could unveil antibiotic genes in the gut bacteria, and might lead to advances in understanding and treating pathogens, and give instructions of adjusting gut flora composition.
By sequencing of single molecules directly, third-generation sequencing technologies have the capability to sequence much longer reads than next-generation sequencing, creating a renaissance in high-quality genome sequencing, allowing for unbiased, more specific identification and even greater taxonomic resolution than second-generation sequencing methodologies for gut microbiota studies.
DNA microarray (also known as gene chip) is a technology widely used in microbial communities profiling, in a DNA microarray, a series of sequences or genes are orderly arranged to detect target DNAs or RNAs (most commonly as cDNA). The basic principle of DNA microarray is nucleic acid hybridization, the location and sequence of each spot of the slide is recorded and compared to get the needed information. This technique enables researchers to investigate and analyze the expression of thousands of genes in a single reaction and address issues more straightforwardly in both prokaryotic and eukaryotic cells.
Virome investigations in intestinal samples by high-throughput sequencing assays are much less conducted compared to bacteriomes because of their lower proportion and the absence of pan-viral sequence signatures. One of the most important features of viruses is the highly variable genomes, with no gene regions shared by all viruses that can be utilized as the universal amplification primers. Until the Wide-scale application of next-generation sequencing makes it possible to study virome at a high-throughput level. Viral metagenomic sequencing is the acknowledged approach for studying the virus. With the development and maturation of NGS technologies, viral metagenomic sequencing has been used successfully to study viruses in the gut community with stool samples and has identified many novel viruses in clinical samples.
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