16S Metagenomic Sequencing of Cecal Microbiome

Total genomic DNA was extracted from the cecal content using the QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. The extracted bacterial DNA concentration was determined using NanoDrop one (Thermo Fisher Scientific, Madison, WI) and stored at -20°C until further analysis. The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified as outlined in Illumina 16S Metagenomic Sequencing Library guideline (Illumina) with the following modifications: Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Life Technologies Corporation, Grand Island, NY) was used to set up the PCR reaction, and Mag-Bind Total Pure NGS beads (Omega Bio-Tek) were used for PCR Clean-Ups, and 35 cycles were used in the PCR. Briefly, 16S rRNA sequencing involved the following steps: 1) 1^st stage PCR: Amplicon PCR, 2) PCR Clean-Up, 3) 2^nd Stage PCR: Index PCR, 4) 2^nd PCR Clean-Up 5) Library quantification, normalization, and pooling, and 6) Library denaturing and MiSeq sample loading. The specific sequence of 16S rRNA gene used for amplified were forward primer (5′-CCTACGGGNGGCWGCAG-3′) and reverse primer (5′GACTACHVGGGTATCTAATCC- 3′) [16 (link)]. Finally, amplicons were normalized and pooled, and subsequently sequenced on the Illumina MiSeq sequencer at the University of Hawaii at Manoa in Genomics, Proteomics, and Bioinformatics core facility.