Microbiome Sequencing and Bioinformatic Processing
Individual swab DNA extracts were barcoded, pooled and sequenced on the
Illumina MiSeq platform (250 bp paired-end reads) in two assays: (1)
barcoded 16S primers (515F and 806R) (38) were used to examine bacterial
diversity; and (2) barcoded 18S v4 primers (TAReuk454FWD1 and
TAReukREV3) (39) were used to examine microeukaryote diversity. 16S
libraries were constructed at the Universidade Estadual Paulista (BR)
and sequenced at the Tufts University Core facility (USA) while 18S
library preparation and sequencing was performed at the University of
Michigan (USA). Negative (template-free) controls were run
simultaneously with each sequencing library to ensure there was no
contamination from PCR or sequencing reagents.
Sequences were quality-filtered and processed using the Quantitative
Insights into Microbial Ecology (QIIME) MiSeq pipeline using default
settings (40). As no mock community was included as a positive
sequencing control, low abundance OTUs were filtered from the dataset
using a conservative abundance threshold (<0.005% of all
reads) (41). Sequences were clustered into operational taxonomic units
(OTUs) using a 97% similarity threshold and compared against reference
databases (rdp GreenGenes for 16S, Silva 97 for 18S) to assign taxonomy
using the BLAST search algorithm. Chimeras were identified and filtered
using UCHIME2 (42). For 18S data, sequences assigned to frog or other
non-target non-microbial species (e.g. , Streptophyta) were
filtered from the dataset. Sequences were rarefied to 1000 per sample
for 18S data and 2000 per sample for 16S data. These values were
selected based on visual examination of histograms and read accumulation
curves constructed for all samples.
To infer ecological effects of bacteria on fungi and protists, bacterial
OTU representative sequences from the T. taophora samples were
compared against a reference database containing bacteria that were
previously isolated from amphibian skin and evaluated for effects onBd growth in co-culture experiments (43). The BLAST algorithm was
implemented and an E-value threshold of E < 1e-20 was used to
identify OTU matches with the reference database. Matching T.
taophora skin bacteria were categorized as Bd enhancing,Bd inhibiting, or having no effect on Bd growth.