DOI: 10.5061/dryad.nk98sf827
title: ASV output data for all in the details: A first assessment for the viability of metabarcoding in diet composition analysis of African wild dogs (Lycaon pictus)

description: DNA metabarcoding is an emerging contemporary technique in diet
 composition studies and stands to fill key knowledge gaps left by
 traditional diet analysis methods. For endangered species such as the
 African wild dog, the fulfilment of these knowledge gaps presents an
 opportunity for improved management practices and vulnerability
 assessments. There are an estimated ~600 African wild dogs remaining in
 South Africa. These dogs are generally understood to prey upon impala
 Aepyceros melampus and other medium-sized ungulates. Here, we present the
 first assessment of DNA metabarcoding as a valuable method for diet
 composition analysis of this highly social carnivore. DNA from faecal
 samples collected across seven landscape types in the Kruger National Park
 (KNP) was extracted and used to determine the presence of seven unique
 prey taxa, including novel species such as the Cape hare Lepus capensis.
 Impala was identified as a prey item in all landscape types, which
 complements the diet preference prediction made with SIA using the same
 samples and the existing understanding of wild dog diet. Given recommended
 improvements, the application of DNA metabarcoding in wild dog diet
 analysis shows promising prospects for identifying novel prey species and
 validating previous records of this endangered canids diet.
description: <em>Sample collection</em>
 Fourteen faecal samples were collected across seven landscape
 types of the Kruger National Park and initially stored at -20°C at the
 SANParks Veterinary Wildlife Services Biobank. They were released to
 Crossey and colleagues at the time of their study in 2021 after being
 cleared as negative for tuberculosis. Samples were lyophilized from
 frozen, ground and sieved through a 20µm metal mesh strainer in sterile
 laboratory conditions. This was done to remove undigested material from
 the faecal matter. <em>DNA extraction and
 sequencing</em> DNA extractions were conducted
 with 40 mg of faecal sample added to 140 µl of water in a 2 ml
 Eppendorf<sup>®</sup> (Merck) tube. Extractions were conducted
 for 14 samples using the DNeasy Blood & Tissue Extraction Kit
 (QIAGEN) with modifications. Modifications included a one-minute vortex
 step before a one-minute centrifugation step at 14000 rpm following the
 three-hour incubation step at 56°C after the addition of proteinase K
 (Inqaba Biotech). Following this, the supernatant was used according to
 the standard kit protocol. The remaining solid faecal sample was stored
 and used for re-extraction as required. Re-extraction was used for nine of
 the 14 samples, and the same protocol as described above was followed,
 with repeat additions of reagents and an additional three-hour incubation
 step. Universal vertebrate primers (Anatech) that amplified the 12S and
 16S gene regions of the mitochondrial genome, designed by Wang<em>,
 et al.</em>(Table 3), were used. The primers were designed to
 amplify a wider set of vertebrate species than previous commonly used
 primers. These primers were degenerate, meaning some positions in the
 sequence have several possible bases labelled according to the standard
 IUPAC nucleotide code. For the polymerase chain reaction (PCR, Saiki
 <em>et al</em>. 1988), both forward primers for the 12S gene
 fragment (VertU V12S-U F1 and Vert U V12S-U F2) were added in equal
 volumes. Each amplification reaction was conducted in a total reaction
 volume of 25µl. Each 25µl reaction included: 3.5 mM
 MgCl<sub>2</sub>, 1 x reaction buffer, 0.25mM of each of the
 four deoxyribonucleotide bases (Inqaba Biotech), 0.16 µM each of the 12S
 or 16S-forward and reverse primers, 0.75 U of
 SuperTherm<sup>®</sup> Taq DNA Polymerase (ThermoFisher
 Scientific), 8 U of BSA (bovine serum albumin, Inqaba Biotech) and 4µl of
 DNA. PCR thermocycling was conducted in a 2720 Thermal Cycler (Applied
 Biosystems). Polymerase chain reaction cycles consisted of an initial
 denaturation phase that separated the strands of existing DNA and lasted
 for 2 min at 94°C. This was followed by 35 cycles of denaturation,
 annealing, and elongation. The denaturation phase lasted for 30s at 94°C,
 the annealing phase, where primers bind to the existing template DNA
 strands, continued for a 20s period at 47°C, then the elongation phase
 proceeded for 20s at 72°C to allow the primers to extend and synthesise
 the new DNA strands. Finally, an extended elongation phase lasted for 5
 min at 72°C to ensure complete synthesis of all fragments. PCR
 amplification success was assessed through electrophoresis using 1.5%
 agarose (Separations) gels using 4 µl of the PCR product. Negative
 controls, using distilled water in place of DNA, showed no contamination.
 To confirm that the PCR reagents were functional, DNA extracted from the
 blood sample of a female lion <em>Panthera leo,</em> was used
 as the positive control. Replicate PCR reactions were performed, the
 number of which depended on the PCR amplification success of each reaction
 for each sample (between 3 - 5 repeats per sample were performed and later
 combined to increase the concentration of the amplified product to an
 acceptable level for downstream applications). After amplification, the
 PCR products were purified according to the protocol outlined in the
 IonXpress<sup>TM </sup>Plus gDNA Fragment Library Preparation
 user guide (ThermoFisher Scientific). PCR products were purified using
 1.8X sample volume of Agencourt™ AMPure™ XP Reagent (Beckman Coulter).
 Following purification, the concentration of each DNA sample was measured
 using Qubit Fluorometric Quantification according to the protocol outlined
 in the Qubit™ dsDNA HS Assay Kit user guide (ThermoFisher Scientific) and
 using three microliters of DNA in each sample reading. Replicates were
 combined such that each sample had a minimum concentration of at least 100
 ng/µl of DNA. The eleven samples were not pooled into a single library but
 were kept separate. Each sample comprised a combination of the 12S and 16S
 purified products, combined in equal concentrations. Library construction
 and sequencing were conducted by the Central Analytical Facilities,
 Stellenbosch University, and the Ion Torrent Next Generation Sequencing
 platform was  used for sequencing.
 <em>Metabarcoding bioinformatics</em>
 The raw sequenced reads first underwent pre-processing to filter
 and correct any errors introduced during amplification and sequencing.
 Pre-processing was conducted using the shell script bbduk.sh in Ubuntu,
 part of the BBTools package. The reads were also trimmed to remove
 low-quality positions based on their Phred score. Primer sequences were
 removed, and sequences were filtered to a minimum of 80bp. Processing and
 post-processing were then conducted in R using the open-source software
 package DADA2. Sequences were further filtered in R to be a minimum of
 150bp long. During processing, Amplicon Sequence Variants (ASVs) were
 clustered by grouping sequences based on minimal nucleotide differences,
 frequently one single nucleotide difference. DADA2 generated a parametric
 error model trained on each sequencing run to minimise and correct false
 positive errors, and effectively collapse the sequences into ASVs.
 Following clustering, taxonomic identification of ASVs was conducted using
 a similarity-based method that aligns query sequences with a chosen
 reference database. A custom reference database was generated, comprising
 47 likely prey species from Kruger National Park compiled using sequences
 from annotated whole mitochondrial genomes present on GenBank (Appendix,
 Table A1). The custom database was generated based on existing knowledge
 of the diet of the African wild dog, prey species known to inhabit the
 Kruger National Park, and preliminary BLAST searches conducted on GenBank.
 Taxonomic assignment was accomplished using the R package taxonomizr,
 which efficiently assigned taxonomy and accession numbers to taxonomic IDs
 by providing functions that inspect NCBI taxonomy files and accession
 dumps. The R package Phyloseq was used to perform further analyses, remove
 overrepresented ASVs and ASVs with less than 10 reads, and construct a
 taxonomy table. Identifications with a percent identity of less than 95%
 were excluded from further analyses.
description: # ASV output data for wild dog diet composition analysis by metabarcoding
 Contained in this data file are the output ASV sequences and their
 taxonomic matches produced using the DADA2 pipeline in R. These are the
 results of DNA extraction from 11 samples of wild dog faeces across
 landscape types in the Kruger National Park. One sheet contains all
 sequences produced by the pipeline, while another sheet shows the
 resultant sequences remaining after filtering the dataset for percent
 identity (pid) matches greater than 95% and removing contaminant DNA.
 Sample landscape types are also given. ## Description of the data and file
 structure The data is provided in an excel format with percent
 compositions of each identified species given for each sample as well as
 information on the quality of the taxonomic match provided. NA indicates
 no species match. These can be used to estimate species contributions to
 diet in each landscape type according to which samples fall in each
 landscape type. Version 2 was uploaded as the previous version was
 realised to be uploaded from an earlier version of the results which
 omitted the Swainson's francolin (*Pternistis Swainsonii*)
 identification. ## Sharing/Access information No other sources to share
 from where this data was derived from.