zOTU-Table_V1V3_Auto-FMT
The excel sheet contains zOTU Counts, annotated taxonomy, sample mapping, confidence intervals for the taxonomy and zOTU sequences.
16S rRNA sequencing
Samples were prepared for sequencing at the sequencing core facilities in Freising (Core Facility Microbiome; ZIEL - Institute for Food & Health) and Regensburg (Institute for Clinical Microbiology and Hygiene, core facility microbiome). Microbial DNA was isolated from about 150 mg of each fecal sample by bead beating, followed by purification using guanidinium thiocyanate and N-lauroylsarcosine to remove cellular components and polyvinylpyrrolidone to remove phenolics as well as cleaning of the DNA with RNase A and the NucleoSpin gDNA Clean-up Kit (Machery-Nagel) (Freising core facility). In Regensburg, microbial DNA was isolated by bead beating on a TissueLyzer II instrument (Qiagen) followed by purification of stool lysates by the MagNA Pure 96 system (Roche) (Regensburg core facility). Bacterial 16S rDNA copy numbers were quantified from extracted DNA as described before. Microbiome sequencing was conducted in Regensburg with a DIN EN ISO 15189 accredited workflow. Briefly, the V1–V3 variable regions of the 16S rRNA gene were amplified in each sample using universal primers S-D-Bact-0008-c-S-20 and S-D-Bact-0517-a-A-18 and resulting amplicons were sequenced on an Ion GeneStudio S5 plus instrument (Thermo Fisher Scientific). Raw sequencing data were retrieved from TorrentSuite 5.18 and further subjected to cutadapt 4.1 78 for adapter and primer removal, demultiplexing and Trimmomatic 0.4 79 for sliding-window based quality filtering. Raw sequencing data were submitted to the European Nucleotide Archive (ENA) and are available under project accession number PRJEB55998.
Analysis of bacterial composition (From PMID 32619440)
Sequencing data were preprocessed using a VSEARCH 2.21.1-based 80 pipeline. Reads with an expected error rate of above 5 were removed. Zero-radius OTUs (zOTUs) were built from quality-filtered reads applying an alpha value of 2 and a minimum size of 5 reads. Chimeric sequences were removed using the uchime3_denovo algorithm. Filtered reads with 98 percent pairwise identities were mapped back to non-chimeric zOTUs with the usearch_global algorithm. Taxonomy was assigned in R version 4.1.3 using the IDTAXA algorithm of DECIPHER 2.22 81 and the The All-Species Living Tree project database version 01.2022.
Calculation of diversity indices and statistical analysis
Diversity analysis was performed with mia using the default parameters. Shannon effective number of species was calculated to assess alpha diversity. Differences between groups were tested for significance using the Wilcoxon signed-rank test. Beta diversity was assessed by unweighted UniFrac distances. PERMANOVA analysis was used to evaluate the significance between groups. Results were considered significant for p ≤ 0.05. A (PCoA) plot based on unweighted UniFrac matrices was constructed to demonstrate the overall dissimilarity of bacterial communities between study participants. Heatmap of the 40 most abundant genera was generated with pheatmap 1.0.12 after centered-log and Z-transformation of zOTU counts. Features and samples were hierarchically clustered by the complete linkage method.
