2.5 DNA extraction and sequencing
Based on the HMP standard operating protocol, the microbial DNA was extracted from fecal samples using the PowerSoil isolation kit (Mo Bio Laboratories, Carlsbad, CA, USA) at Environmental Microbial Genomics Laboratory in Georgia Institute of Technology. The 16S rRNA amplicon libraries were prepared for the 16S rRNA V4 region31,32. These 16S rRNA amplicons were generated using KAPA HiFi HotStart ReadyMix (KAPA Biosystems, KK2600) and primers specific to 16S V4 region of Bacteria and indices were attached using the Nextera XT Index kit (Illumina, FC-131-1001). Clean-up was performed on the indexed libraries using AMPure XP beads. The 16S libraries were pooled in equal amounts based on fluorescence quantification. Each run included a control template to test for PCR accuracy and possible contamination. Final library pools were quantitated via qPCR (Kapa Biosystems, catalog KK4824). The pooled library was sequenced on an Illumina miSeq using miSeq v3 600 cycle chemistry (Illumina, catalog MS-102-3003) at a loading density of 8 pM with 20% PhiX, at PE300 reads. The microbial sequencing led to paired-end sequences.