DOI: 10.5061/dryad.2jm63xszk
title: Data from: To pool or not to pool: Pooled metabarcoding does not affect estimates of prey diversity in spider gut content analysis

description: Gut content metabarcoding has provided important insights into the food
 web ecology of spiders, the most dominant terrestrial arthropod predators.
 In small invertebrates, like spiders, gut content analysis is often
 performed on whole body DNA extracts of individual predators, from which
 prey sequences are selectively enriched and sequenced. Since many spider
 species are generalist predators, large numbers of samples comprising
 individual spider specimens must be analyzed to recover an exhaustive
 image of a spider species’ prey spectrum, which is costly and
 time-consuming. Pooled processing of bulk samples of multiple
 specimens has been suggested to reduce the necessary workload and cost
 while still recovering a representative estimate of the prey diversity.
 However, it is still unclear if pooling approaches lead to bias in
 recovering the prey spectrum and if the results are comparable to data
 from individually processed spiders. Here, we test the effect of
 metabarcoding pooled spider gut content on the recovered taxonomic
 diversity and composition of prey. Using a newly adapted primer pair,
 which efficiently enriches COI barcode sequences of diverse arthropod prey
 groups while suppressing spider amplification, we test if pooling leads to
 reduced taxonomic diversity or skewed estimates of prey composition. Our
 results show that pooling and individual processing recover highly
 correlated taxonomic diversity and composition of prey. The only exception
 are very rare prey items which were less well recovered by pooling. Our
 results support pooling as a cost effective and time efficient approach to
 recover the diet of generalist predators for population-level studies of
 spider trophic interactions.
description: Individual spiders were collected using beat sheets, sweep nets,
 and hand sampling between June and July 2021 at a grassland site in
 Kimmlingen, Rhineland-Palatinate, Germany (49°49'58.4"N
 6°36'05.8"E). Immediately after collection, all specimens were
 separated into individual tubes with pure ethanol and stored at room
 temperature. In the lab they were morphologically identified to species
 level, measured for body size (prosoma width), and stored at -20°C. Adult
 males were excluded from further analysis due to their reduced feeding
 activity (Pollard et al., 1995), as were specimens with visible damages to
 minimize the risk of contamination from external DNA fragments.
 DNA was extracted from each spider individually. Prior to DNA
 extraction, all samples were treated with 0.15% bleach (NaOCl) for 30
 minutes according to Greenstone et al. (2012) to remove external DNA
 contamination. Since entire bodies were used for DNA extraction, they were
 homogenized for 30 seconds at maximum speed (SPEX 1600MiniG, Metuchen, New
 Jersey, USA), each with two sterile stainless steel beads in 600µl lysis
 buffer with 3µl Proteinase K (Invitrogen, Waltham, United States). Cell
 lysis was then performed at 55°C for 16 hours. Subsequent DNA extraction
 used the Qiagen Puregene Kit and followed the manufacturer's protocol
 (Qiagen, Hilden, Germany). GlycoBlue (Invitrogen, Waltham, United States)
 was added as coprecipitant (1:600) during the DNA precipitation step to
 visualize DNA and maximize its yield.  To compare the
 performance of pooling and individual processing of gut content samples,
 we generated pools of DNA from a diverse set of spider species. We
 prepared eight pools, each consisting of equal volumes of ten DNA extracts
 from different spiders. Four pools were species-specific and comprised ten
 individuals of one species each (either <em>Agelena labyrinthica,
 Evarcha arcuata, Mangora acalypha</em> or <em>Synema
 globosum</em>). The other four pools were composed of ten
 individuals that belonged to different species, but with approximately
 similar size (XS = 0.5-1.0mm, S = 1.5-2.0mm, M = 2.5-3.0mm, L = 3.0-3.5mm
 prosoma width, Supplemental table 1). These DNA pools will be referred to
 as “Pool” hereafter. Each of the spiders in one pool were also amplified
 and sequenced individually. After DNA sequencing, the same combinations of
 samples as before were merged computationally (see Fig. 1B, Supplemental
 table 1), hence creating an exact individually processed replicate of the
 pooled sample. These computationally merged pools will be referred to as
 “Reference Pool” hereafter.  Please note that we chose
 to use pools from extracted DNA rather than to pool spiders before DNA
 isolation. The latter approach would have made a comparison of the
 recovered prey richness and composition with individually processed
 spiders impossible. By pooling DNA extracts, and still processing the same
 extracts individually, an exact comparison of the effect of pooling on
 patterns of prey diversity can be observed. The incentive of this study
 however, is to provide insight into the suitability of extracting DNA from
 bulk samples instead of individual extractions. PCRs
 were performed using the Qiagen Multiplex PCR Kit in 10 µl volumes with 1
 µl DNA and 0.5 µl of each 10 µM primer in 5µl Multiplex and 3µl RNAse free
 water. The PCR amplification was performed in two rounds. The first round
 consisted of an initial denaturation at 95°C for 15 min, and 32 cycles
 with an annealing temperature of 45°C (and additionally increments up to
 50°C in the gradient PCR) for 90s and extension at 72°C for 90s, omitting
 final elongation. This PCR used the new primers with 20bp long tails added
 to the 5’-end as templates for the following indexing PCR. This indexing
 PCR consisted of 5 cycles of the same protocol as before, but with 56°C
 annealing temperature to introduce the Illumina TruSeq adapters and dual
 indices. Amplification success of each PCR step was verified on a 2%
 agarose gel stained with GelRed. Amplicons were combined into the final
 library using approximately equal amounts of DNA, depending on their band
 intensity on the agarose gel. Final libraries were purified with 1:1
 AmPURE XP beads (Beckman and Coulter, California, USA) and sequenced in
 multiple runs on an Illumina Miseq platform with V3 chemistry 300 cycles.
 To control for contamination, blank extractions and blank PCRs were
 included in each respective batch and sequenced alongside the experimental
 samples. Data analysis  Reads were
 demultiplexed using CASAVA (Illumina, San Diego, CA, USA) and allowing no
 mismatches in indices. The demultiplexed reads were then merged using PEAR
 (Zhang et al., 2014) with a minimum overlap of 50bp and a quality
 threshold of 20. The resulting merged reads were quality-filtered for at
 least 90% of bases exceeding Q30, and then converted to FASTA files using
 the FastX toolkit (Gordon and Hannon, 2010). Valid sequences were selected
 by retaining only sequences beginning with the forward primer and ending
 with the reverse primer, allowing for variation only in degenerate sites
 of the primer sequences. Primer sequences were then trimmed with
 <em>sed</em> in UNIX. Reads were
 dereplicated using USEARCH (Edgar, 2010) and the dereplicated sequences
 were clustered into zero-radius OTUs (zOTUs) using the
 <em>unoise3</em> command (Edgar, 2016) with <em>de
 novo</em> chimera removal. Taxonomic identity was assigned to zOTU
 sequences using BLASTn (Altschul et al., 1990) against the complete NCBI
 nucleotide database (downloaded 12/2022), with the top 10 hits retained. A
 custom Python script (Schoeneberg, 2023) assigned taxonomy from the BLAST
 output. Sequences with non-arthropod hits among the top ten BLAST hits
 were excluded from further analyses. For all others, the first hit was
 used for zOTU annotation. This resulted in an OTU table consisting only of
 zOTUs belonging to Arthropoda. Annotated zOTUs were then filtered to a
 minimal percent identity of 90% and a minimal fragment length of
 60bp.
description: # Data from: To pool or not to pool: Pooled metabarcoding does not affect
 estimates of prey diversity in spider gut content analysis
 [https://doi.org/10.5061/dryad.2jm63xszk](https://doi.org/10.5061/dryad.2jm63xszk) ### Files and variables #### File: raw\_sequences.zip **Description**: This folder consists of 150 fastaq.gz for different spider species. The raw sequence data from Illumina sequencing in fastq format. #### File: metadata.csv **Description:** metadata of the samples ##### Variables * *sample_id* : unique sample identifier, (naming scheme corresponding only to collector, not any other quality of the sample) Note that some single specimen samples have been used in multiple DNA pools and therefore occur multiple times in this list, but with different pool IDs and rarefaction depths.   - *type*: sample type (single specimen sample = sample consisting of the DNA extracted from a singular spider individual; DNA pool = sample consisting of multiple "single specimen samples" combined) * *pool_id*: name of the pool sample each sample belongs to. Note that some single specimen samples have been used in multiple DNA pools and therefore occur multiple times in this list, but with different pool IDs and rarefaction depths.  - *species*: spider species of the sample; diverse= pool mixed from diverse species, see associated individual samples for details * size_mm: prosoma width in mm; NA = not applicable to pools, as they contain multiple spider individuals with varying sizes - *prey_reads*: sum of reads obtained from the respective sample that belong to Arthropoda, but not Araneae zOTUs; * *rarefied_to*: rarefaction depth. Note that some single specimen samples have been used in multiple DNA pools and therefore occur multiple times in this list, but with different rarefaction depths depending on the pool.  #### File: tax.csv **Description:** taxonomic annotation of the quality-filtered zOTUs **Variables** * otu_id: unique identifier of each zero-radius OTU * perc: similarity to the NCBI database hit * len: length of the matching sequence between query sequence and NCBI sequence * phylum, class, subclass, order, superfamily, genus, species: taxonomic annotation of each otu_id from the NCBI database * missing values identifier: NA