DOI: 10.5061/dryad.4qrfj6qfz
title: Data from: Are fecal samples an appropriate proxy for amphibian intestinal microbiota?

description: The intestinal microbiota, an invisible organ supporting a host’s
 survival, has essential roles in metabolism, immunity, growth, and
 development. Since intestinal microbiota influences a host’s biology,
 application of such data to wildlife conservation has gained interest.
 There are standard protocols for studying the human intestinal microbiota,
 but no equivalent for wildlife. A major challenge is sampling the
 intestinal microbiota in an effective, unbiased way. Fecal samples are a
 popular proxy for intestinal microbiota because collection is
 non-invasive, convenient, and allows for longitudinal sampling. Yet, it is
 unclear whether the fecal microbiota is representative of the intestinal
 microbiota. In amphibians, research on sampling methodology is limited. In
 this study, we characterize and compare microbiota (small intestine, large
 intestine, feces) of two Hong Kong stream-dwelling frog species: Lesser
 Spiny Frog (Quasipaa exilispinosa), and Hong Kong Cascade Frog (Amolops
 hongkongensis). We found that both species have similar dominant phyla and
 families, but diverge in terms of the dominant genera. Next, we assess the
 performance of fecal microbiota in representing the intestinal microbiota
 in these two species. We found that (1) microbiota of small and large
 intestine differs significantly, (2) feces are not an appropriate proxy of
 both intestinal sections, and (3) a set of microbial taxa significantly
 differs between sample types. Our cautions equating fecal and intestinal
 microbiota. Sampling feces can avoid sacrifice of an animal, but
 researchers should avoid over-extrapolation and interpret results
 carefully.
description: <strong>Frog
 surveys</strong> Night surveys were conducted within three hours
 after sunset, from June to August 2021. Each survey included 3–4 surveyors
 experienced in herpetological field work. Surveyors walked along the
 stream looking for the two target species using headlamps and flashlights.
 Individuals were captured with gloved hands, with gloves changed between
 individuals to prevent cross contamination. After sampling skin microbiota
 for another study, each individual was stored in separate air-filled
 plastic bags for transport to laboratory within two
 hours. <strong>Fecal microbiota
 </strong> All equipment was sterilized with a 10% bleach
 solution beforehand to avoid contamination. Upon arrival to the
 laboratory, each individual was kept separately in sterile plastic boxes
 for 12–24 hours waiting for defecation. The boxes have a wire mesh floor
 that allows the feces to fall and avoid contamination by the individual.
 Each box was humidified by spraying sterile water, and kept in dark to
 minimize disturbance. Approximately nine hours after capture, boxes were
 checked hourly for defecation. No food was provided during the process.
 Fecal samples were collected using sterile forceps and stored in tubes at
 –80 °C until DNA extraction. Individuals were euthanized for sampling
 intestinal microbiota (see below) after defecation, or after 24 hours
 without defecation. <strong>Intestinal
 microbiota</strong> Each individual was euthanized by overdosing
 with ethyl 3-aminobenzoate methanesulfonate in the laboratory (MS222;
 Sigma-Aldrich, St. Louis, MO, USA) (American Veterinary Medical
 Association, 2020). The entire intestine was dissected from end of the
 stomach to the cloaca. The small and large intestine were identified
 morphologically. A 2-cm piece of anterior and posterior end of the
 intestine was taken as small and large intestine, respectively (Tong et
 al., 2014). We could not identify intestinal subsections morphologically
 (e.g., duodenum, jejunum, and ileum of the small intestine), so this
 information was excluded from our analyses. Each segment was rinsed to
 remove transient bacteria and intestinal content with 2 mL sterile water
 using a pipette. Each segment was then cut longitudinally on one side,
 laid flat, and the gut microbiota was collected by swabbing using an
 ESwabTM (Copan Diagnostic Inc., California, USA). The swab was drawn
 across the inner surface of the intestine segment 10 times (1 time =
 forward and backward). The swabs were stored at –80 °C until DNA
 extraction. For a negative control, a blank swab was included in
 subsequent DNA extraction and sequencing. Frog bodies were preserved and
 accessioned into the herpetology specimen collection of the Lingnan
 University Natural History Collection.
 <strong>DNA
 extraction</strong> DNA extraction was conducted at the laboratory
 of Lingnan University. DNA was extracted from swabs using the
 QIAamp®BiOstic® Bacteremia DNA Kit (Qiagen GmbH, Hilden, Germany)
 following amendments verified by the manufacturer. The amendments are
 summarized as follows—in the first step, 450 µL of MBL buffer was added to
 each PowerBead tube: (1) swabs were defrosted then placed in PowerBead
 tubes, (2) feces were subsampled (250 mg) and placed in PowerBead tubes,
 and (3) the subsequent steps followed the manufacturer’s protocol starting
 from “vortex for 10 s and incubation at 70 °C for 15 min”. A blank sample
 was included for every batch of DNA extraction as a negative control. DNA
 extractions were quantified by Qubit™ 3 Fluorometer with the dsDNA BR
 Assay Kit (Thermo Fisher Scientific Inc., MA, USA).
 <strong>Library
 preparation and sequencing</strong>
 Sequencing libraries were
 prepared according to the Illumina protocol (“16S Metagenomic Sequencing
 Library Preparation Part #15044223 Rev. B protocol"). The V4 region
 of the 16S rRNA gene was amplified by PCR using The Earth Microbiome
 Project V4 primer pair coupled with Illumina adapter overhang sequences
 (515F [Parada]: 5’-
 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA-3’, 806R [Apprill]:
 5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNVGGGTWTCTAAT-3’) (Caporaso
 et al., 2011; Apprill et al., 2015; Parada et al., 2016). For each
 reaction, 2 ng of DNA was amplified with 5X reaction buffer, 1 mM of dNTP
 mix, 500 nM of each PCR primer, and Herculase II fusion DNA polymerase
 (Agilent Technologies, Santa Clara, CA). Thermal cycling conditions were
 as follows: 3 min at 95 °C; 25 cycles of 30 s at 95 °C, 30 s at 55 °C, and
 30 s at 72 °C; followed by 5 min at 72 °C. The 1st PCR product was
 purified with AMPure beads (Agencourt Bioscience, Beverly, MA). For each
 sample, 2 µL of the 1st PCR product was amplified for library construction
 using a unique Nextera XT Index Primer (Illumina, Inc., San Diego, CA).
 Thermal cycling conditions were the same as the 1st PCR, but with only 10
 cycles. The 2nd PCR product was then purified using AMPure beads
 (Agencourt Bioscience, Beverly, MA), and quantified using two approaches:
 (1) qPCR according to the qPCR Quantification Protocol Guide (KAPA Library
 Quantification kit for Illumina Sequencing platforms) and (2) TapeStation
 system and D1000 ScreenTape (Agilent Technologies, Waldbronn, Germany).
 The pooled products were paired-end sequenced with Herculase II Fusion DNA
 Polymerase Nextera XT Index V2 Kit on an Illumina MiSeq PE300 (Illumina,
 Inc., San Diego, CA). A negative control was included in each lane to test
 for contamination. All library preparation and sequencing work was
 conducted at Macrogen (Seoul, South Korea).
description: Bioinformatics work was
 performed using Quantitative Insights into Microbial Ecology 2 (QIIME 2,
 version 2022.11) (Bolyen et al., 2019) and R (R Core Team 2021). Raw
 sequence data were demultiplexed by Macrogen (Seoul, South Korea). We
 evaluated the quality of demultiplexed reads and trimmed to remove
 poor-quality bases. Sequence quality control and denoising were performed
 with DADA2 (Callahan et al., 2016) to generate amplicon sequence variants
 (ASVs) using q2-dada2. Representative sequences were aligned with MAFFT
 (Katoh and Standley, 2013) using q2-alignment. To maximize taxonomic
 classification accuracy, we assigned taxonomy to ASVs using a weighted
 Bayes classifier that incorporates information on environment-specific
 taxonomic abundance (Kaehler et al. 2019), pre-trained on the 16S
 515F/806R region in SILVA 138 (Quast et al., 2013) (MD5:
 b9476399080d189b4c9917d1246e7c69) (Bokulich et al., 2018; Robeson II et
 al., 2021) using q2-feature-classifier (Bokulich et al., 2018). Sequences
 were removed if unassigned at the domain level or assigned to be
 mitochondria/chloroplast/archaea. The format of the metadata file used in
 QIIME2 was validated by the cloud-based Google Sheets add-on Keemei
 (Rideout et al., 2016). The output QIIME2 files were exported to R for
 subsequent analysis using R package “qiime2R” (Bisanz
 2018).
description: # Are fecal samples an appropriate proxy for amphibian intestinal
 microbiota? This study compared the bacterial community of three sample
 types (feces, small intestine, large intestine) on two stream-dwelling
 amphibian species (*Amolops hongkongensis*, and *Quasipaa exilispinosa*)
 sampled in Hong Kong. The dataset is based on metabarcoding of the 16SV4
 region on the Illumina MiSeq PE300 platform. We showed that feces is not
 an approxiate proxy of both intestine sections, and identified a set of
 microbial taxa significantly differs between sample types. ## Description
 of the Data and file structure This dataset contains compressed text-based
 format in .fastq.gz format to store nucleotide sequence and its
 corresponding quality scores in both sequencing directions. Please refer
 to the metadata file for more details. The file name contains information
 as follows: Take "AMHO-LR-LI01_1.fastq.gz" as an example, 1. The
 first section "AMHO" indicates species. Two available options:
 "AMHO": *Amolops hongkongensis* (Hong Kong Cascade Frog)
 "QUEX": *Quasipaa exilispinosa* (Lesser Spiny Frog) 2. The
 second section "LR" indicates sampling location. Seven available
 options: "TMS": Sze Lok Yuen, Tai Mo Shan Country Park
 "MTL": Mui Tsz Lam, Ma On Shan Country Park "MTL2":
 Tai Shui Hang, Ma On Shan Country Park "LR": Lion Rock Country
 Park "TT": Tai Tam Country Park "LAN": Tei Tong Tsai,
 Lantau Country Park "TL": Ting Kau, Tai Lam Country Park 3. The
 thrid section "LI01" indicates sample type, and individual
 number Three available options for sample type, no limit for individual
 number "SK": Skin microbiota "SI": Small intestinal
 microbiota "LI": Large intestinal microbiota 4. The fourth
 section indicates sequencing direction Two available options
 "1": Forward reads "2": Reverse reads In this study,
 the dataset was imported into Quantitative Insights into Microbial Ecology
 2 (QIIME 2, version 2022.11) (Bolyen et al., 2019) and R (R Core Team
 2021) for sequence filtering, denoising, and subsequent analysis. ###
 Reference Bolyen, E., Rideout, J. R., Dillon, M. R., Bokulich, N. A.,
 Abnet, C. C., Al-Ghalith, G., et al. (2019). Reproducible, interactive,
 scalable and extensible microbiome data science using QIIME 2. Nature
 Biotechnology, 37(8), 852857.
 R Core Team. (2021). R:
 A language and environment for statistical computing. R foundation for
 statistical computing. Vienna, Austria.