Bacterial DNA extraction and sequencing
We extracted total DNA from feces using a ZymoBIONICS DNA kit and sent
DNA extractions to the University of Connecticut Microbial Analysis,
Resources and Services for sequencing with an Illumina MiSeq platform
and v2 2x250 base pair kit (Illumina, Inc.). We also sequenced a
laboratory extraction blank to control for kit contamination. We
conducted bacterial inventories via amplification of the V4 region of
the 16S rRNA gene using primers 515F and 806R and with Illumina adapters
and dual indices (Kozich et al., 2013). We used the DADA2 (v. 1.22.0)
pipeline (Callahan et al., 2016) in R (v. 4.2.0) to process sequence
data. After quality assessment, we trimmed sequences to remove low
quality read areas and chimeric reads. We classified amplicon sequence
variant (ASV) taxonomies using RDP’s Naive Bayesian Classifier (Q. Wang
et al., 2007) with the Silva reference database (v. 138.1) (Quast et
al., 2012). After classification, we removed sequences identified as
chloroplast and mitochondria from the dataset. We identified and removed
likely bacterial contaminants with the package decontam (Davis et
al., 2018) in R using the kit extraction blank that was processed in
parallel with the other samples as a control. Sequences were aligned
using the DECIPHER package (v. 2.22.0) in R (Wright, 2015), and a
generalized time-reversible maximum likelihood tree of the remaining
ASVs was constructed with the phangorn package version 2.9.0
(Schliep, 2011). The ASV table, taxonomic information, phylogeny, and
sample metadata were joined for bacterial community analyses using the
package phyloseq (McMurdie & Holmes, 2013). We filtered the
feature table to retain samples with at least 1,500 total reads which
reduced the dataset to 58 samples containing 2,526 unique ASVs. The
resulting data set had an average of 51,056 ± 6,178 reads per sample
(min: 1,732; max: 294,720). For alpha and beta diversity analyses, we
rarefied samples to the sample with the lowest read count (1,732). The
filtered dataset contained 1,448 ASVs after random subsampling.