Participants received OMNIgene•GUT stool and OMNIgene•ORAL saliva collection kits, with standardized collection instructions. They were instructed to refrain from eating, drinking, chewing gum, smoking, or brushing teeth 30 minutes before saliva collection. Kits contained reagents to rapidly homogenize samples at the point of collection and maintain the DNA stable at ambient temperature for 60 days, with no cold chain required. Participants were instructed to return their samples in prepaid mailing boxes within 2 weeks of collection.
DNA extraction, sequencing, and annotation were performed by Diversigen (Minnesota, USA). Upon receipt of the samples, visual observations of the collection kits were recorded, including sample discoloration, low volume, and incorrect usage. Additional quality controls included determining that there were at least 16,000 16S copies/μL and greater than 1ng/µL of DNA per sample. Microbial DNA was extracted at room temperature from saliva and stool samples using the QIAGEN Powersoil Pro DNA isolation kit with a liquid-handling robot, and according to standardized protocols (QIAGEN, Netherlands). Sequence libraries were prepared using enzymatic tagmentation with low cycle polymerase chain reaction. 88 samples plus a positive and negative library prep control were included in each 96 well-plate. Sequencing was performed with the BoosterShot® shallow shotgun sequencing platform, with 1x100 reads at a target depth of 2 million reads per sample. The Cutadapt software tool was used to discard sequences with an average Q score less than 30. Additionally, samples with fewer than 10,000 reads were discarded.