## 3 Microbiome Analysis by Culture-Independent Methods
### High Through-Put Sequencing  
The culture-independent methods based on PCR amplification of specific target genes, commonly the 16S rRNA gene, which serves as an evolutionary chronometer and allows phylogenetic analysis of obtained sequences from the microbial community [77], are becoming a routine technique used with a sharp increase of its acceptance today. Initially, the DNA-based techniques were applied to single colonies or libraries of PCR-amplified nucleotide sequences from environmental samples, after successful cloning of the single PCR products, but in recent years, the application of next generation sequencing (NGS) technology to microbial communities has revolutionized research on ecological microbiology, enabling, at an affordable cost, both deep information and also large-scale sequencing of DNA samples without culturing and cloning. This simplifies the analytical procedures involved and also makes this technology accessible based on paid-for-service. Metagenomics for the characterization of microbial community phylogeny and diversity is a new line of molecular microbiological methods based on genomic DNA extraction directly from environmental samples and then NGS technology to obtain the short reads of nucleotides for further assembly and analysis [78; 79; 80]. Metagenomic technologies can be either target-specific or non-specific. For the target-specific ones, the microbial community of bacteria and archaea is revealed based on the 16S rRNA gene, and that of eukaryotes based on the 18S rRNA or additional genes. [PERSON] et al. [31] investigated the bio-erosion of the sandstone of the Royal Palace of Angkor Thom and the temple Beng Mealea using the NGS targeting the 16S rRNA gene with samples of biofilm and exfoliated materials to obtain phylogeny and diversity information of the bacterial community in them. The results of this study using the adhesive sampling technique revealed that a higher bacterial diversity was detected in exfoliated sandstone materials than in the biofilms, but the diversity of bacteria was lowest in the lower layers of the biofilm after repetitively sampling at a selective location (discussed above). Firmicutes and Gemmatimonadetes were the dominant phyla and were detected only in exfoliated materials, while Cyanobacteria, Chloroflexi, and unassigned bacteria were more abundant in the biofilms. The results of this study suggested a microbial specificity on the sandstone monuments by the nature of the sample types and the different degree of deterioration. Biofilms grown on the surface of sandstone are in constant contact with the underlying inorganic substratum, the shortage of organic nutrients resulting in lower diversity of the community of heterotrophic bacteria adhering on the sandstone surface. Moreover, Cyanobacteria and Chloriflexi were identified as the key photoautotrophic microorganisms colonizing the surface of the sandstone. They appeared widely in the biofilms of Angkor monuments with a strong ability for utilizing direct or indirect sunlight, resistance to the high temperature, and the ability to survive the hypersaline environment of sandstone niche [33; 38; 56; 62; 81]. The active metabolism of these autotrophic microorganisms results in the fixation of the inorganic CO\({}_{2}\) as a carbon source and the ability to modify the surface layer for retention of water, providing opportunities for subsequent colonization by the heterotrophic bacteria and fungi [38; 40; 56; 58; 59; 82]. Then, a diverse and stable microbial community can be formed on the exfoliation sandstone materials, and the microbial activity and abundant metabolites further accelerate the attack of the sandstone. Although the target-specific methods can amplify and sequence marker regions of the ribosomal DNA to obtain a genus-level picture of the microorganisms present in different environmental samples, the targeted metagenomic analysis alone could not yield enough information about the genes and the biochemical functions in a microbial community because of the lower depth of sequencing [83].