Traditional culture based and phenotypic methods are time consuming and fail to identify unculturable or unusual species. The use of 16S ribosomal RNA in bacterial taxonomic classification is widely recognized in clinical practice and research, especially in the study of bacteria with unusual phenotypic profiles, rare bacteria, slow–‐growing bacteria, uncultivable bacteria and culture–‐negative infections.
The 16S rRNA gene sequence was first used in 1985 for phylogenetic analysis. Because it contains both highly conserved regions for primer design and hypervariable regions to identify phylogenetic characteristics of microorganisms, the 16S rRNA gene sequence became the most widely used marker gene for profiling bacterial communities. Limited by sequencing technology, the 16S rRNA gene sequences used in most studies are partial sequences. Therefore, the selection of proper primers is critical to study bacterial phylogeny in various environments. Primers are designed to recognize and bind some specific and restricted target sequences of the bacterial 16S rRNA gene sequence, known as the 9 “hypervariable regions” (V1-V9) that can provide species specific signatures.
The advent of NGS has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. We are equipped with the Ion Torrent™ Personal Genome Machine System (PGM), one of the most used and standardized platform for metagenomics analysis of different microbiota.
The PGM system is a next generation sequencing (NGS) method able to analyze mixed microbial populations in a culture free environment. PGM system perform direct sequencing of samples, quickly and with more amplicons, to enable better discrimination between organisms. By amplifying and sequencing seven hypervariable regions (V2, V3, V4, V6, V7,V8 and V9) of bacterial 16S rRNA, PGM system allows an accurate detection and identification of a broad range of bacteria down to genus or species level. DNA is extracted from biological samples and the bacterial DNA is amplified using the Ion 16S.