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).