4. Variable region of the 16S rRNA gene

The 16S rRNA gene sequence was first used in 1985 for phylogenetic analysis [30]. 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 [31]. Full-length 16S rRNA gene sequences consist of nine hypervariable regions that are separated by nine highly conserved regions [32]. Limited by sequencing technology, the 16S rRNA gene sequences used in most studies are partial sequences. Therefore, the selection of proper primers is critical for studying bacterial phylogeny in various environments [32].

Recent studies utilizing high-throughput technology also have demonstrated that the use of suboptimal primer pairs results in the uneven amplification of certain species, causing either an under- or overestimation of some species in a microbial community [32, 33]. Integrated bioinformatics tools were used to evaluate the phylogenetic sensitivity of the hypervariable regions compared with the corresponding full-length sequences. Results showed that using a combination of V4–V6 regions represented the optimal subregions for bacterial phylogenetic studies of new phyla [34].
