16S rRNA Amplicon Sequence of Turtle Microbiome
We performed DNA extractions using the DNeasy Blood & Tissue kit (Qiagen N.V., Hilden, Germany) following the manufacturers protocol. We processed both types of samples equally, except that we ran the proteinase K digestion step on swabs for one hour and on scrapes for eight hours. Following DNA extraction, we diluted each sample by a factor of 1:10 using sterile water. We amplified the bacterial 16S rRNA V4-region using the amplifying primer pair F515/R806. We ran each sample three times and each PCR reaction consisted of 3.0 L of template DNA, 7.5 L of 2X HotStart Master Mix (New England Biolabs Inc., Ipswich, MA), 1.0 L of 1 nM forward and reverse primers, and 3.5 L of sterile water for a total of 15 L per reaction. Our first PCR conditions consisted of 94 C for 3 min, 30 cycles of 94 C for 45 s, 50 C for 60 s, and 72 C for 90 s, followed by 72 C for 10 min. We pooled amplicon triplicates and cleaned them using the GeneJet PCR Purification kit (ThermoFisher Scientific Inc., Whaltman, MA) following the manufacturer's protocol. We implemented a second PCR on the clean PCR products to ligate dual-index barcodes attached to Illumina sequencing adaptors to the ends of amplicons (Hernandez-Gomez, et al., 2017). Our second PCR consisted of 5.0 L of clean amplicons, 7.5 L 2X HotStart Master Mix, 1.0 L of 1 nM forward and reverse barcode primers, and 1.5 L of water for a total of 15 L reactions. PCR conditions consisted of 94 C for 3 min, 5 cycles of 94 C for 45 s, 65 C for 60 s, and 72 C for 90 s, followed by 72 C for 10 min. We quantified the second PCR using a High-Specificity Qubit Fluorometer assay (ThermoFisher Scientific Inc., Whaltman, MA), pooled them in equimolar amounts, and cleaned the pool using the GeneJet PCR Purification kit. The sample pool was shipped on dry ice overnight via the United States Postal Service to the Cornell University Institute of Biotechnology for sequencing using a 250 bp paired-end kit on a MiSeq DNA machine (Illumina Inc, Carlsbad, CA).