16S rRNA gene profiling

16S rRNA gene profiling is a one of the most widely used methods to determine the taxonomic distribution within bacterial communities, like the intestinal microbiome. The 16S rRNA gene, which codes for a component of the bacterial ribosome, contains highly conserved and highly variable regions. The highly variable regions can be used to determine taxonomy.

16S rRna analysis
Created in BioRender.com and modified by Sven Haange
Using primers which bind to the highly conserved regions in vicinity of the variable regions of the 16S rRNA gene, the adjacent variably region is amplified by PCR in all bacteria of the a sample. Then, the amplicons are sequenced by next generation sequencing. The raw sequencing data is processed by bioinformatics to determine amplicon sequencing variants (ASVs) and assign the resulting reads. The sequneces of the ASVs are compared to data bases containing 16S rRNA gene sequences of known bacteria to determine taxonomy. The relative distribution of taxons can be determined by the number of sequencing reads assigned to each ASV.
In our group, we have workflows to extract and purify DNA from fecal samples as well as to process raw sequencing data using the Dada2 algorithm on the Galaxy server at the Helmholtz Centre for Environmental Research. In addition, we have an in-house pipeline based on R-scripts for data analysis of the processed 16S rRNA gene sequencing data for biological and statistical analysis.