*2.5. MiSeq Pipeline Method*

The processing of the raw reads started with a quality check and filtering of the low quality (<Q25) reads using Trimmomatic 0.32. After passing the QC check, the pairedend sequence data were merged using PandaSeq. The primers were then trimmed with ChunLab's in-house program at a similarity cutoff of 0.8. The noise was removed from the sequences using Mothur's pre-clustering program, which merges the sequences and extracts unique sequences allowing up to two differences between the sequences. The Ez-Taxon database was used for taxonomic assignments using BLAST 2.2.22 and pairwise alignment was used to calculate similarity. Uchime and the non-chimeric 16S rRNA database from EzTaxon were used to detect chimerism in the reads with best hit similarity rates below 97%. Sequence data were then clustered using CD-Hit and UCLUST, and alpha diversity analysis was conducted.
