Gut microbiome of Gopherus polyphemus
The DNA samples were prepared for targeted sequencing using the Quick-16S Plus NGS Library Prep Kit and custom-designed primers from Zymo Research. The V3-V4 primer set was utilized in this project. PCR reactions were performed in real-time PCR machines to limit chimera formation. The final PCR products were quantified, pooled based on equal molarity, and cleaned up with Select-a-Size DNA Clean & Concentrator. The final pooled library was quantified using TapeStation and Qubit. Control samples, including positive and negative controls, were used for DNA extraction and targeted library preparation. The final library was sequenced on an Illumina MiSeq with a v3 reagent kit and 10% PhiX spike-in. The calculated total DNA was determined using genome size assumptions (four 16S genes per genome) and Avogadro's number.