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.