16S rRNA gene sequences of avian cloacal microbiome
Cloacal swabs were used to collect gut microbiome samples from wild Hawaii amakihi and warbling white-eyes from Hawaii Island. DNA was isolated from the cloacal swabs and from negative control swabs using the QIAamp PowerFecal Pro DNA Kit, then quantified using the Qubit dsDNA HS Assay Kit and Qubit 2.0 Fluorometer. DNA libraries were prepared for 16S rRNA sequencing by amplifying the V3V5 regions of the 16S rRNA gene using the 16S Illumina Amplicon Protocol and universal primers 515F (GTGCCAGCMGCCGCGGTAA) and 939R (CTTGTGCGGGCCCCCGTCAATTC). Index PCR was performed on the amplicons to attach custom i5 and i7 adaptors that provided each cloacal sample a unique sequence barcode identity. The BluePippin protocol, trained on 500640 bp fragment collection for size selection, was used to remove off-target sequences. Qubit was used to quantify the final pooled samples and prepare 4 nM DNA libraries that were sequenced on a single Illumina MiSeq run using MiSeq Reagent Kit v3 with a 25% PhiX spike.