16S of duodenal mucosa: adult human
Mucosal biopsies underwent DNA isolation using a Qiagen MagAttract Powermag microbiome DNA isolation kit (Germantown, MD). Barcoded dual-index primers specific to the V4 region of the 16S rRNA gene were used to amplify DNA, using a touchdown PCR protocol given that duodenal mucosa is potentially low biomass.[8] Multiple negative controls were run in parallel. Microbiome sequencing occurred in three separate runs. Libraries were prepared and sequenced using the Illumina 500-cycle MiSeq reagent kit V2 (San Diego, CA).[9]