DNA Extraction, PCR, and Sequencing
We extracted microbial DNA from perianal swabs using a modified Qiagen
DNeasy PowerSoil HTP 96 Kit protocol (Qiagen Inc., Maryland, USA). We
isolated swab tips using scissors sterilized by dunking blades in 80%
ethanol solution before passing them through a flame. We randomized
samples from different islands and species across PowerBead plates to
minimize potential batch effects, and disrupted all samples and controls
using a MiniBeadBeater-96. We shook plates for 3min, allowed 30sec of
rest, rotated plates, and shook plates for an additional 3min. We then
followed the standard manufacturer protocol before eluting samples for
15min at room temperature in 60μL solution C6 pre-heated to 70ºC.
We used polymerase chain reaction (PCR) to amplify and tag the 16S rRNA
hypervariable 4 (V4) region with the barcoded forward
(GTGCCAGCMGCCGCGGTAA) and reverse (TAATCTWTGGGVHCATCAGG) primers
described in . PCR reactions contained 5μL HiFi HotStart ReadyMix (KAPA
Biosystems, Massachusetts, USA), 3.2μL primer mix (1.25μM), and 2.0μL
template DNA. Extraction controls included sterile swab tips (negative
controls) and DNA extracted from ZymoBIOMICS Microbial Community
Standard (D6300; positive controls); and PCR controls included PCR
reagents with no template DNA added (negative controls) and 1μL of
ZymoBIOMICS Microbial Community DNA Standard (D6305; positive controls;
Zymo Research, California, USA). PCR cycling conditions included:
initial denaturation of 94ºC/3min, touchdown cycling for 30 cycles of
[94ºC/45s, 80-50ºC/60s, 72ºC/90s] decreasing 1ºC each cycle, 12
cycles of [94ºC/45s, 50ºC/60s, 72ºC/90s], and a final extension of
72ºC/10min. To maximize yield and minimize PCR artifacts, each sample
was run through PCR in duplicate and subsequently combined for
quantification using a Qubit Flex Fluorometer (Invitrogen,
Massachusetts, USA). We then pooled 40ng of each barcoded library, used
the QIAquick PCR Purification kit (Qiagen Inc., Maryland, USA) and E-Gel
2% Size Selection Protocol (Invitrogen, Massachusetts, USA) to clean
and size select libraries, and subsequently used Agencourt AMPure XP
magnetic beads (Beckman Coulter, California, USA) to concentrate final
libraries using a 1.25X bead to library ratio. Libraries were paired-end
sequenced (2x150nt) across three sequencing plates on an Illumina MiSeq
in the Princeton University Lewis Sigler Genomics Core Facility.