| | | Using high-throughput sequencing of 16S ribosomal genes, the composition of microbial samples can be characterized to an extent that one would never have dreamed of a decade ago. The multiplexing of pyrosequencing by DNA-barcoding techniques further allows the simultaneous characterization of hundreds of samples at low cost. This approach provides the unique opportunity to gain deep insights into the ecology of microbes, to monitor community dynamics over time, or to study the effects of environmental perturbations on community structures. However, despite the wide application of high-throughput sequencing of 16S genes, there is no consensus about the "optimal" primer pairs for an unbiased amplification and the "optimal" variable region to be targeted. A validation study is presented, demonstrating that the choice of PCR primers and targeted 16S region can strongly affect the obtained results and biological conclusions. Furthermore, the computational characterization of the generated data sets is discussed. Finally, the power of high-throughput 16S DNA sequencing is high-lighted on a real-world example investigating the composition of microbial samples from the gastrointestinal tract. |
