16S rRNA gene amplicon sequencing of layer hen ileal microbiota
DNA from ileal luminal content samples was extracted using the Qiagen Powerlyzer soil kit. Samples were then sequenced using 250 bp paired-end reads for each sample of the V4 region of the 16S rRNA gene (515F, 806R; Caporaso et al., 2011; Caporaso et al., 2012) at the Iowa State University DNA Facility using Illumina MiSeq sequencing technology.