Amplicon sequencing is frequently used to identify and differentiate microbial species. Short less than 500 bp hypervariable regions of conserved genes or intergenic regions are amplified by PCR and analyzed using NGS technology, and the resulting sequences are compared against microbial databases.

For bacteria and archaea, the 16S rRNA gene is the most common target for amplicon sequencing. For fungi, three targets are generally used: the 18S rRNA gene and two internal transcribed spacers (ITS) located between rRNA genes. These regions are usually sufficiently divergent to separate even highly related species, and can sometimes differentiate subspecies.

At Genetikode, we have sequenced over 170,000 microbial samples for our customers. Our standard bioinformatics analyses include alpha-diversity analysis, OTU analysis, species annotation, beta-diversity analysis, and multi-variate statistical analysis. Applications range from identifying a single species in pure culture to characterizing the microbiota of animals or plants, to comparing species diversity and population structure in various environmental sources or geographic regions. Our specialists can advise you on the appropriate analyses for your project.

• Data quality control.
• OTUs (Operational Taxonomic Units) clustering and filtering.
• Alpha-diversity analysis, including rarefaction curve, Chao-1 curve, Shannon curve, rank abundance curve, and alpha indices table.
• OTUs analysis and species annotation, phylogenetic composition analysis, phylogenetic tree, heatmap, and taxonomic tree.
• Beta-diversity analysis, including unweighted UniFrac distance heatmap, PCA (principal component analysis), PCoA (principal co-ordinates analysis), UPGMA (unweighted pair-group method with arithmetic means), and NMDS (Non-metric multidimensional scaling) analysis.
• Multi-variate statistical analysis, including LEfSe (LDA effect size) analysis, metastats analysis, ANOSIM, and MRPP analysis.