DOI: 10.5061/dryad.2v6wwpzpc
title: DNA metabarcoding data characterizing insectivorous diet of purple martins (Progne subis subis) using two COI primer sets (ANML and ZBJ)

description: DNA metabarcoding is a molecular technique frequently used to characterize
 diet composition of insectivorous birds. However, results are sensitive to
 methodological decisions made during sample processing, with primer
 selection being one of the most critical. The most frequently used DNA
 metabarcoding primer set for avian insectivores is ZBJ. However, recent
 studies have found that ZBJ produces significant biases in prey
 classification that likely influence our understanding of foraging
 ecology. A new primer set, ANML, has shown promise for characterizing
 insectivorous bat diets with fewer taxonomic biases than ZBJ, but ANML is
 not yet widely used to study insectivorous birds. Here, we evaluate the
 ANML primer set for use in metabarcoding of avian insectivore diets
 through comparison with the more commonly used ZBJ primer set. Fecal
 samples were collected from both adult and nestling Purple Martins (Progne
 subis subis) at two sites in the USA and one site in Canada to maximize
 variation in diet composition and to determine if primer selection impacts
 our understanding of diet variation among sites. In total, we detected 71
 arthropod prey species, 39 families, and 10 orders. Of these, 40 species
 were uniquely detected by ANML, whereas only 11 were uniquely detected by
 ZBJ. We were able to classify 54.8% of exact sequence variants from ANML
 libraries to species compared to 33.3% from ZBJ libraries. We found that
 ANML outperformed ZBJ for PCR efficacy, taxonomic coverage, and
 specificity of classification, but that using both primer sets together
 produced the most comprehensive characterizations of diet composition.
 Significant variation in both alpha- and beta-diversity between sites was
 found using each primer set separately and in combination. To our
 knowledge, this is the first published metabarcoding study to directly
 compare avian diet characterizations produced with both ANML and ZBJ
 primer sets.
description: Data represent DNA metabarcoding data produced with two COI
 primer sets (ZBJ and ANML) applied to DNA extracts from purple martin
 (<em>Progne subis subis</em>) fecal samples. Whole fecal
 samples were collected opportunistically from nestling Purple Martins
 during nest checks and banding activities in Orlando (FL, USA n = 68),
 Erie (PA, USA n = 19), and Winnipeg (Manitoba, Canada, n = 36) during the
 2019 breeding season. Whole fecal samples or
 sub-samples of feces collected with sterile swabs were collected from
 adults at the Orlando site only (n = 11) during this same time
 period. Genomic DNA was extracted from a homogenized subsample of each
 fecal sample using the DNeasy PowerSoil kit. Library
 preparation consisted of (a) amplicon PCR (50 cycles for ZBJ, 40 cycles
 for ANML), and (b) indexing PCR (8 cycles) using the Illumina XT kit. PCR
 reactions were cleaned up with 1.1X Sera-Mag SpeedBeads, and PCR2 products
 (i.e., libraries) were quantified spectrophotometrically before equimolar
 pooling. The final library pooled was QC checked by TapeStation and
 quantified by qPCR. The library pool was
 sequenced using a paired-end approach (2x300bp) with v3 sequencing
 reagents (Illumina, MS-102-3003) following manufacturer’s instructions,
 with the addition of a final 2-minute denaturing step at 96°C. The library
 pool was spiked with 20% PhiX (Illumina, FC-110-3001) and loaded onto the
 flow cell at 15 nM. DNA sequencing reads were
 demultiplexed on the MiSeq instrument into separate FastQ files for each
 sample amplified with either ZBJ or ANML barcoding primer sets. DNA
 sequencing data are published in raw form as fastq files; no read
 processing has been performed on these files.