DNA extractions for 16s sequencing

On week five of tadpole development, we humanely euthanized N = 6 (Experiment 1) and N = 10 (Experiment 2) tadpoles per treatment group for 16S rRNA targeted amplicon microbiome sequencing analysis. We randomly selected tadpoles from each tank and weighed tadpoles to the nearest 0.1 g and measured snout-to-vent length (hereafter, SVL) to the nearest 0.1 mm to calculate body condition (mass/SVL). We euthanized the tadpoles using sterile MS-222 that we neutralized by adding sodium bicarbonate to pH 7.0. We then transferred the tadpoles to Powerbead Pro Tubes (Qiagen, Valencia, CA, USA). We homogenized the tadpoles using a tissue homogenizer (Mixer Mill MM 400; Retsch, Newtown, PA, USA) for 3 minutes, each at 25 hz. We then extracted DNA from the homogenized samples using the QIAmp PowerFecal Pro DNA Kit (Qiagen, Valencia, CA, USA). We included N = 4 water blanks for Experiment 1 and N = 5 water blanks for Experiment 2 for which we followed the same protocol for extraction, substituting molecular-grade water instead of a tadpole sample. We then shipped the samples on ice to the Idaho State University Molecular Research Core Facility for 16S rRNA sequencing. 

We received fastq files from Idaho State University that were demultiplexed and had primers/adapters removed. These fastq files have been uploaded.

Development and Exposure Experiment with Batracochytrium dendrobatidis (Bd)

We collected skin swab samples to test for Bd presence and infection intensity using standardized swabbing techniques. During the infection experiment, we continued to collect mass, SVL (as described above), and skin swab samples for diagnostic testing every two weeks until the termination of the experiment. We extracted DNA from our swabs using the DNeasy Blood and Tissue DNA Extraction Kit (Qiagen, Valencia, California, USA). We then quantified Bd DNA on the swabs using real-time quantitative polymerase chain reaction (qPCR). We used an internal positive control and a dilution set of plasmid standard (Pisces Molecular, Boulder, CO, USA) to quantify Bd load. We determined Bd load by using the cycle threshold (Ct) value to calculate genomic equivalences. We also adjusted the Bd plasmid copy numbers by accounting for dilution during the extraction process.  

The raw sequencing data files are uploaded in a .fastq file format. All other data files are uploaded as Excel (.xlsx) files. Please see README files for more information.