Dolphin Oral Microbiome Sequencing

Fifty-four dolphin oral samples were selected for 16S rRNA gene amplicon sequencing. Genomic DNA was extracted from dolphin oral samples and 32 negative (PBS) controls using the DNeasy UltraClean 96 Microbial Kit (Qiagen Cat. #10196-4) following manufacturer’s instructions. The 16S rRNA V4 region was amplified using 515F and 806rB primers using Platinum™ II HotStart PCR Master Mix (ThermoFisher Cat. #14000013). The PCR products were pooled at equal volume and gel-purified. Final purification was performed using Macherey-Nagel NucleoSpin Gel and PCR Clean-up, Mini Kit (Fisher, Cat. #740609). Amplicons were sequenced on the Illumina MiSeq platform with 250-bp paired reads at the Stanford Chan Zuckerberg Biohub Facility, resulting in a median read depth of 92,077 reads (min: 50,772, max: 265,108).
Demultiplexing was performed using Bcl2Fastq v. 2 (Illumina, CA, USA). ASVs were inferred using DADA2^{71 (link)} v. 1.16.0, following guidelines in the “Big Data Workflow” (https://benjjneb.github.io/dada2/bigdata_paired.html). Taxonomic affiliations were assigned using the SILVA 138 SSU database^{72 (link)} as a reference. Forward and reverse reads were trimmed to 240 and 180 nt, respectively. This pipeline yielded a total of 1116 taxa and 5,339,751 reads across the 54 samples. ASVs were analyzed using phyloseq v. 1.28.0^{72 (link)}.