16S of V4: mouse stool
Template DNA quantity in samples was determined by creating 1:8 and 1:64 dilutions of each sample and subjecting them all to qPCR using primers V4_515F_Nextera and V4_806R_Nextera targeting the V4 locus in the rRNA gene. Samples were diluted with water to normalize starting template abundances, and then amplicons were generated by subjecting sample DNA to 25-cycle PCRs using the same primers. PCR products were diluted 1:100 in water and subjected to a second 10-cycle PCR to attach Illumina sequencing primer-compatible DNA regions as well as individual barcodes for each sample. Samples were all uniquely dual-indexed, as detailed in Gohl, et al. (Gohl, et al., 2018). Final PCR products were normalized using SequalPrep kits (Invitrogen), pooled into sequencing libraries, and cleaned with AMPure XP mag beads (Beckman Coulter). Sequencing libraries were loaded onto an Illumina MiSeq using a 2x300 v3 flow cell (Illumina, San Diego, CA).