16S rRNA Amplicon Sequencing

The 16S rRNA amplicon sequencing technique is a microbiome analysis where different samples are analyzed at the same time using multiplexing. The results can be used to evaluate microbial diversity at genus, family, order, class, and phylum levels. The resolution is normally insufficient to evaluate the species level. The different steps in the bioinformatical analysis allow both the analysis of all samples combined or comparisons between samples. The bioinformatics analysis focuses on quality control of reads, merging of identical reads and grouping of reads into operational taxonomic units (OTUs) with a threshold of 97%. The threshold is inherited from the species threshold for classification of species based on 16S rRNA sequence comparison. The distribution of reads and OTUs within and between samples can be used to estimate α- and β-diversity, respectively. An alternative to OTU dereplication is divisive amplicon denoising algorithm (DADA). This algorithm groups reads according to statistical modeling identifying a most probable central sequence, and Amplicon sequence variants (ASVs) is the term. The relative occurrence of the taxonomic units at the levels of genus, family, order, class, and phylum can be compared between samples. These distributions can be related to metadata by principal component analysis.