Sampling and Sequencing
Sampling was conducted as previously described
(Lutz et al., 2015).
Briefly, birds were collected using a combination of mist-netting and
ballistic capture in 2009 and 2011. Blood samples were obtained via
brachial venipuncture; oral (buccal) and cloaca samples were collected
by swabbing with sterile cotton swabs; liver, spleen, and intestinal
tracts were dissected from euthanized animals. All samples were stored
in cryogenic vials and flash-frozen in liquid nitrogen until nucleic
acid extraction. Samples collected for this project were processed
following the Earth Microbiome Project standard processing protocols
(Thompson et al., 2017).
Briefly, DNA was extracted using 96-well PowerSoil PowerMag DNA
extraction plates (Qiagen), which were homogenized using a TissueLyser
beadbeater (Qiagen). From eluted DNA, triplicate PCRs using the
515f/806r EMP primers amplified the V4 region of the 16S rRNA gene,
pooled amplicons were sequenced on Illumina MiSeq and HiSeq instruments,
and data were uploaded to the Qiita web-based microbiome analysis
platform for initial processing (made available previously via QIITA ID
11166 by (Song et al.,
2020). All sequences were demultiplexed and quality filtered using the
Qiime2 pipeline (Bolyen et
al., 2019) and forward and reverse reads were joined and processed
using Deblur to remove sequencing errors and refine sequences to
amplicon sequence variants (ASVs)
(Amir et al., 2017).
Deblurred ASV tables and sample metadata were further processed using
Qiime2 (Bolyen et al.,
2019). To assess the presence of artefactual batch effects associated
with microbiota diversity, statistical significance of the plate on
which the samples were placed on were calculated on each sample type
using the adonis2 function (PERMANOVA) from the vegan package using
unweighted UniFrac and weighted UniFrac
(McArdle & Anderson, 2001);
(Oksanen et al., 2018). Only
the unweighted UniFrac distances of the intestine samples showed
significant (p <0.05) differences due to plate
(platename R2= 1.714%, p = 0.05).