16S rRNA sequencing of human mucosal swabs (rectal, nasal)
DNA and RNA were extracted from nasal and rectal swabs using the Qiagen AllPrep PowerViral DNA/RNA extraction kit. Bacterial template DNA quantity was determined by performing qPCR using the V3_357F_Nextera and V4_806R_Nextera primers targeting the V3V4 locus of the rRNA gene. Samples were diluted with water if necessary, and amplicons were generated using a 25-cycle PCR reaction using the same V3V4 primers. PCR products were diluted in water and subjected to a second 10-cycle PCR reaction to attach Illumina sequencing primer-compatible DNA regions as well as individual barcodes for each sample. The samples were all uniquely dual-indexed. Final PCR products were normalized using SequalPrep kits, pooled into sequencing libraries, and cleaned with AMPure XP mag beads. Sequencing libraries were loaded onto an Illumina MiSeq using a 2x300 v3 flow cell