Intestinal Microbiome Amplicon Sequencing

The intestinal biopsy samples were prepared for 16S sequencing using our Microbiome Amplicon Sequencing Workflow [35 (link)]. Briefly, the pre-extracted DNA [17 (link)] was first amplified in duplicate using dual-indexing Illumina primers (forward: ACGCGHNRAACCTTACC; reverse: ACGGGCRGTGWGTRCAA) that targeted the V6-V8 region (438 bp) of the bacterial 16S rRNA gene. The pooled duplicate PCR products were verified using high-throughput E-gels (Invitrogen), then purified and normalized using the SequalPrep 96-well Plate Kit (Invitrogen). Following quantification, the pooled samples were run on an Illumina MiSeq using PE 300 + 300 bp v3 chemistry at the Integrated Microbiome Resource (Dalhousie University, Halifax, Nova Scotia).