Environmental DNA metabarcoding differentiates between micro-habitats within the rocky intertidal

While the utility of environmental DNA (eDNA) metabarcoding surveys for biodiversity monitoring continues to be demonstrated, the spatial and temporal variability of eDNA, and thus the limits of the differentiability of an eDNA signal, remains under-characterized. In this study, we collected eDNA samples from distinct micro-habitats (~40 m apart) in a rocky intertidal ecosystem over their exposure period in a tidal cycle. During this period, the micro-habitats transitioned from being interconnected, to physically isolated, to interconnected again. Using a well-established eukaryotic (cytochrome oxidase subunit I) metabarcoding assay, we detected 415 species across 28 phyla. Across a variety of univariate and multivariate analyses, using exclusively taxonomically assigned data as well as all detected amplicon sequence variants (ASVs), we identified unique eDNA signals from the different micro-habitats sampled. This difference paralleled expected ecological gradients and increased as the ..., 1.1.  Reproducibility
To improve reproducibility (e.g. Dickie et al., 2018; Shea et al., 2023), enable open data science (e.g. Fredston & Lowndes, 2024), and aid in the initiation of new eDNA biomonitoring projects, we have published detailed, step-by-step protocols for many of the methods described, including specific materials used, photographs, and additional methodological notes not possible to include here. See Shea and Boehm for sample collection and filtering (2023b), for DNA extractions (2023c), for PCR amplification (2023d), and shipping samples (2023e). Additionally, we have published all data and code for replicating our analyses via Dryad, including FASTQ files and eDNA datasets (pre-processing & post-processing). Through Dryad and Zenodo, our modified Anacapa Container and scripts for bioinformatics (Shea & Boehm, 2023a), as well as a GitHub repository including an R Markdown file that reproduces all methods & results (Shea & Boehm, 2023f) can also be ac..., , # Environmental DNA metabarcoding differentiates between micro-habitats within the rocky intertidal

This repository includes the necessary data to process marine eDNA metabarcoding samples collected from Pillar Point, Half Moon Bay, CA, USA on 28 January 2022.

## Description of Data, Code, & File Structure

### FASTQ\_Files.zip

This archive contains compressed FASTQ sequencing files generated from an Illumina MiSeq PE 2x250bp (500 cycles) sequencing run. More details on sequencing methods can be found in the \"Methods\" section on Dryad and the manuscript. The zip file also contains FASTA files of the forward and reverse COI primers used. These FASTA and FASTQ files are formatted for use with the modified Anacapa Container described below.

### Anacapa\_Toolkit\_Output.zip

This archive contains the unprocessed output from a run of the modified Anacapa Container on 31 January 2023 on the FASTQ files contained in FASTQ_Files.zip.

In the archive are three folders:

#### CO1

This fo...