V3 and V4 libraries were sequenced with a MiSeq Reagent Kit v3 (600-cycles) on a MiSeq System (Illumina, San Diego, CA, USA) [29 (link)]. 2x301 cycles of paired-end sequencing were performed, with 5% Phix (Illumina) being added to each library pool.
Operational Taxonomic Unit (OTU) clustering and taxonomic analyses were performed using CLC Genomics Workbench v. 10.1.1 and CLC Microbial Genomics Module v. 2.5 (Qiagen). Sequences were trimmed, merged and clustered into OTUs at 97% sequence similarity with the Amplicon-Based OTU clustering tool. The most abundant sequences were selected as representative of each cluster, and taxonomic levels were assigned using CLC Microbial Genomics default values by comparing against the 2013 Greengenes Database release. Low depth samples (< 9,000 sequences per sample) were removed, and alpha diversity indexes were calculated. The weighted Unifrac metric was employed for the calculation of inter-sample diversity (beta diversity).
Operational Taxonomic Unit (OTU) clustering and taxonomic analyses were performed using CLC Genomics Workbench v. 10.1.1 and CLC Microbial Genomics Module v. 2.5 (Qiagen). Sequences were trimmed, merged and clustered into OTUs at 97% sequence similarity with the Amplicon-Based OTU clustering tool. The most abundant sequences were selected as representative of each cluster, and taxonomic levels were assigned using CLC Microbial Genomics default values by comparing against the 2013 Greengenes Database release. Low depth samples (< 9,000 sequences per sample) were removed, and alpha diversity indexes were calculated. The weighted Unifrac metric was employed for the calculation of inter-sample diversity (beta diversity).
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