To pool or not to pool: Pooled metabarcoding does not affect estimates of prey diversity in spider gut content analysis

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 gu..., 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 (SPE..., , # 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 sa...