Sequence Processing and Analysis
We used a paired-end, dual-indexed barcode splitter implemented inGalaxy to demultiplex raw sequencing data, with allowance for one nucleotide mismatch between expected and observed barcodes. We then imported demultiplexed data into QIIME 2 v2021.11 ; https://qiime2.org) and ran each sequencing plate through the dada2 denoise-pairedplugin to further process reads. This pipeline corrected likely sequencing errors, trimmed low quality bases, merged paired-end reads, and removed chimeric reads. We then combined data from all three sequencing plates into one dataset for further filtering based on reads that appeared in positive and negative control samples.
We included six positive controls to assess possible sources of bias during DNA extraction (n=3, ZymoBIOMICS Catalogue No. D6300) and PCR amplification (n=3, ZymoBIOMICS Catalogue No. D6305). All six samples contained sequences from the three Gram-negative and five Gram-positive bacterial species contained within the mock community standards. This confirmed successful lysis, DNA extraction, amplification, and taxonomic identification of bacteria contained within our dataset. To identify potential contaminants, we implemented the R package decontamusing default parameters for the combined method that considers the frequency (distribution of the frequency of each sequence feature) and prevalence (presence/absence across samples) of potential contaminants, with sequencing plate used as a batch identifier. We subsequently filtered our dataset to remove the 32 amplicon sequence variants (ASVs) identified as possible contaminants by decontam ; 130 ASVs with taxonomic labels of ”mitochondria” or ”chloroplasts”; any ASVs with a total frequency <10; and any ASV found in only one sample. We then removed all positive controls, negative controls, duplicate samples, and samples with fewer than 2500 reads. This produced a final dataset of 3,669,688 sequences and 1,443 ASVs sequenced in 110 samples, including 21 foxes and 22 skunks sampled on Santa Cruz Island, and 33 foxes and 34 skunks sampled on Santa Rosa Island (Table S1).