title: 16S rRNA Seq of salamander skin
design: Microbiota swabs were sent to the Minnesota Genomics and Bioinformatics Center for sequencing. DNA was extracted using the Qiagen DNeasy PowerSoil Pro Extraction Kit. Extracted DNA was amplified for 30 cycles using the Quick-16S Primer Set (515F-806R primers) which target the V4 region of the 16S rRNA gene. PCR products were then sequenced on an Illumina MiSeq using a v3 reagent kit for 600 cycles. FASTQ files returned from the Minnesota Genomics and Bioinformatics Center were analyzed in-house using a prepared Qiime281 pipeline (included in SI). Briefly, reads were filtered and processed using dada2 82 with default trim and length settings changed to: --p-trim-left-f 19, --p-trim-left-r 20,  --p-trunc-len-f 225, --p-trunc-len-r 225. A phylogenetic tree was then created using the Qiime2 align-to-tree-mafft-fasttree function. Next, we trained a nave bayes classifier specific to our primer sets using the feature-classifier Qiime2 function. We then grouped sequenced reads into amplicon sequence variants (ASVs). Finally, ASVs were taxonomically classified using the Silva 99% similarity database and outputted as BIOM files for subsequent analysis.