16S of Centropyge eibli hindgut
Sequencing was performed on an Illumina MiSeq platform in the Trace and Environmental DNA (TrEnD) laboratory at Curtin University in Perth, Australia using 515F (5 GTGBCAGCMGCCGCGGTAA 3) and 806R (5 GGACTACHVGGGTAWTCTAAT 3) primers targeting the V4 region of the 16S rDNA gene. The optimal yield of DNA for PCR reactions was first determined by qPCR, then amplicons were generated using a single round of PCR with fusion tag primers including Illumina adapter regions, unique MID tags, and primers. Quantitative PCR (qPCR) experiments were performed in duplicate on a StepOnePlus Real-Time PCR System using 1x AmpliTaq Gold Buffer, 2mM MgCl2, 0.25 M dNTPs, 10 ug BSA, 1U AmpliTaq Gold DNA polymerase, 2 l template DNA and ultrapure distilled water to 25 l. These reactions were amplified under the following conditions: initial denaturation at 95 C for 5 min, then 35 cycles of 95 C for 30 s, 50 C for 30 s, and 72 C for 45 s, and a final extension at 72 C for 10 min. PCR duplicates of each sample were combined and all samples were then pooled in equimolar ratios. Extraction controls and PCR negative controls containing no sample were added to sequencing runs to assess for cross-contamination. Final sequencing libraries were size selected using a Pippin Prep (Sage Science, Beverly, MA, United States), purified using a QIAquick PCR purification Kit, and sequenced unidirectionally using an Illumina 300 cycle MiSeq v2 Reagent Kit and standard flow cell.