Understanding the diversity of microbial communities, in almost any conceivable environment, has been greatly facilitated by rapid advances in next-generation sequencing (NGS) technologies and associated bioinformatics approaches. 16S rRNA sequencing is a fast, inexpensive research profiling technique based on variation in the bacterial 16S ribosomal RNA (rRNA) gene.
Sequencing the 16S rRNA gene can be used to identify bacterial species, characterize bacteria populations, and perform grouping by shared sequence characteristics (taxonomical assignment). The 16S rRNA gene is highly conserved across domains of Bacteria and Archaea, and taxonomical assignment is possible due to the presence of nine hypervariable regions (V1–V9) that contain sufficient sequence diversity to classify microbes. Moreover, since these variable regions are flanked by conserved regions, PCR amplification using universal primers is possible.
Universal primers targeting the 16S rRNA gene allow a single, or a few, PCR reactions to be used when amplifying bacterial communities as a whole. Due to the clonal nature of massively parallel NGS workflows, each PCR reaction and subsequent sequencing read can be considered to be representative of a single bacterium within a mixed population. This approach has allowed characterization of bacterial communities without isolation and culturing. Through a deep sequencing approach, 16S rRNA research profiling has fundamentally changed our understanding of countless microbial communities and has proven to be an important discovery tool, revealing what is called the “microbial dark matter” of our planet due to the difficulty of culturing most bacterial species.
The Ion PGM™ System has been used to characterize an incredible array of bacterial communities, including microbial populations important for research into human health in diverse sites such as the mouth, airway, complex bacterial infections, diabetic mycetoma (or “Madura foot”), during stool substitute transplants, and the human and murine intestinal tract.
Semiconductor sequencing has also revealed the composition of bacterial populations in a wide variety of environmental sources: such as waterways in response to oil sands mining and sewage; the northern Gulf of Mexico and the English Channel; soil and rhizosphere of a cactus and willow; uranium mine tailings and the tailings-water interface; contaminated arctic soils; aquaculture systems; biowaste reservoirs; bioproduction settings [1, 2, 3]; and during the life cycle of the Lone Star tick.
To learn more about the possibilities of 16S rRNA profiling and bacterial typing research, Dr. Joe Petrosino of Baylor College of Medicine discusses in a series of videos using the Ion PGM™ Sequencer and 16S rRNA sequencing to study microbial associations with Type 1 Diabetes, fecal transplants, and for forensic use, the decomposition of corpses. Here’s a video of David Visi of University of North Texas describing the use of the Ion PGM Sequencer for 16S rRNA analysis of a kenaf microbial retting community (kenaf is valuable fiber plant used in cultivation, and retting is a stage in its processing). Dr. Antonius Suwanto of Bogor Agricultural University in Indonesia) describes his study evaluating the bacterial composition of tempeh samples (a type of soybean) using 16s rRNA sequencing on the Ion PGM™ Sequencer.
See also an application note (PDF) about the collaboration between Dr. George Watts (Genomics Shared Service at the University of Arizona Cancer Center, Tucson, Arizona) and Ion Torrent researchers to optimize the amplicon region targeted in the 16S rRNA gene.