Microbiome Analysis using QIIME2 and Decontamination

Illumina paired-end reads were demultiplexed in the Illumina BaseSpace Sequence Hub [48 ]. The QIIME2 (v. 2021.4) software [49 (link)] was used to process reads, infer the taxonomic affiliation and perform diversity and abundance analysis. The removal of sequence errors, index and adapters was performed with the cutadapt plugin [50 (link)], whereas quality control, chimera removal and dereplication were performed with the DADA2 plugin [51 (link)]. Taxonomic affiliation of amplicon sequence variants (ASVs) was inferred using the Greengenes 13_8 99% classifier [52 (link),53 (link)]. Specifically for the reads assigned to the Acetobacteraceae family, we additionally used the BLASTN [54 (link)] and RDP classifier [55 (link)] for taxonomic identification. The dataset was submitted to a decontamination step using the microDecon package [56 (link)] in R (v. 4.1.1) to remove sequences considered to be cross-contamination (between biological samples) and contaminants from the laboratory environment, human manipulation [45 (link)], DNA extraction kit and PCR reagents (“kitome”) [57 (link)].