WGS of human gut microbiome
For both studies, stool samples were stored in a -20 degree Celsius freezer immediately following collection for up to two weeks, after which aliquots were transferred to longer-term storage in a - 80 degree Celsius ultra-freezer. DNA was extracted using the MoBio PowerFecal DNA Isolation kit. Consistent with our prior study, DNA was diluted for library preparation using the Illumina Nextera XT Library Prep Kit in study 1, and 1 ng from each sample was fragmented and amplified using Illumina Nextera XT Index Kit primers. Amplified DNA was then cleaned using Agencourt AMPure XP beads, resuspended in buffer, and tested again for concentration, quality, and fragment size distribution on a Bioanalyzer using the Agilent High Sensitivity DNA Kit. DNA libraries were brought to the same nM concentration, pooled, and denatured with 0.2 N NaOH prior to loading on an Illumina MiSeq Reagent Kit v3 and sequencing on the Illumina MiSeq platform. For study 2, metagenomic sequencing libraries were prepared using Illuminas Nextera XT library prep kit, and were pooled and sequenced on an Illumina NextSeq 500 machine using a single High-Output v2 Kit (300 cycle, paired-end) at the GWU Milken Institute School of Public Health Genomics Core Facility.