Microbial DNA Extraction and Sequencing from Fecal and Mucosal Samples

We extracted microbial DNA from feces and biopsy (mucosal) samples using MOBIO PowerSoil DNA extraction kit (MOBIO Laboratories, Carlsbald, CA, USA). We prepared sequencing libraries using the protocols from Earth Microbiome project using V4 primers with Illumina Miseq Instrument^{62 (link)}. PANDAseq^{63 (link)} paired reads were analyzed using QIIME 1.9 suite^{34 (link)}. The details of the analysis can be found in the Supplementary Document. Briefly, OTUs were formed at 99% sequence similarity and the OTUs that contained less than 0.005% of the total number of sequences and chimeric sequences were omitted from the analysis as previously recommended⁶⁴ . We calculated alpha and beta diversity metrics of Phylogenetic Diversity Whole Tree^{65 (link)}, and Unifrac^{33 (link)}. Gene abundances for bile acid biosynthesis were predicted with Phylogenetic Investigation of Communities by Reconstruction of Unobserved Species (PICRUSt) software^{53 (link)}. Genus-level phylotypes that significantly differed after RYGB were clustered based on Euclidean distances using ClustVis^{66 (link)}.