16S rRNA Amplicon Sequencing of Microbiome

Community DNA extraction from pooled fecal, manure pack, and Day 84 samples was performed using the QIAGEN DNeasy^® PowerSoil^® Pro kit (Qiagen, Valencia, CA) per the manufacturer’s protocol within the automated QIAcube robot. Library preparation was conducted using Nextera XT Index Kits (Illumina, Inc., San Diego, CA) and Mastercycler^® Thermal Cycler (Eppendorf, Enfield, CT). Amplicon primers 341F (5’-CCTACGGGNGGCWGCAG-3’) and 785R (5′-GACTACHVGGGTATCTA ATCC-3′) with index adapters were utilized to specifically target and amplify the hypervariable V3/V4 region of the bacterial 16S rRNA gene (Klindworth et al., 2013 (link)). Library validation was performed on the Fragment Analyzer System (Agilent Technologies, Inc., Santa Clara, CA) prior to paired-end amplicon sequencing (2 × 300) of the 16S rRNA gene using MiSeq reagent v3 kits and the Illumina MiSeq platform (Illumina, Inc.) per manufacturer’s protocol. The 16S rRNA amplicon sequence data can be found in the NCBI database under BioProject accession number: PRJNA595617.