16SProfilingAnalysis.md

Analysis of 16S profiling data

ASV table generation

Amplicon sequencing reads from A. thaliana roots and Flowpot soil were quality filtered and demultiplexed according to their barcode sequence using QIIME (26) and unique amplicon sequencing variants (ASVs) were inferred from error-corrected reads, followed by chimera filtering. ASVs were mapped to the reference 16S rRNA sequences (downloaded from “www.at-sphere.com”) to generate an ASV count table. All steps were carried out using the Rbec R package (27). Analysis was performed on samples with a sequencing depth of at least 500 high-quality reads.

Alpha- and beta-diversity

Analyses and visualization were performed in the R statistical environment (Version 4.1.2). Alpha and beta diversity were calculated on non-rarefied ASV count tables (28). Alpha-diversity (Shannon index) was calculated with the “plot_richness” function in phyloseq package (29). Beta-diversity (Bray-Curtis dissimilarities) was calculated using the “ordinate” function in phyloseq package and used for unconstrained ordination by Principal Coordinate Analysis (PCoA). Statistical significances were assessed using permutational multi-variate analysis of variance (PERMANOVA) using the adonis function in the vegan package.