Data repository accompanying the paper 'BeeDNA: microfluidic environmental DNA metabarcoding as a tool for connecting plant and pollinator communities' by Harper et al. (2021). 1_Raw_Data.zip This zipped folder contains the raw sequence data (sorted by primer set and demultiplexed) for both sequencing runs (2019-10-24 and 2019-11-11). To decompress each file, run: tar -xvf filename.bz2 This will create a folder for each primer set containing the raw reads for each sample/control. 2_Anacapa_Bioinformatic_Processing.zip This zipped folder contains all files needed to perform bioinformatic processing with Anacapa. Please process sequence data belonging to each primer set individually (i.e. do not process sequence data belonging to different primer sets together). 3_metaBEAT_Bioinformatic_Processing.zip This zipped folder contains the scripts and files needed to perform bioinformatic processing with metaBEAT. Before running the scripts, move the raw reads for each sample belonging to each primer set into the dedicated folder within metaBEAT_Bioinformatic_Processing, e.g. all .fastq files in Raw_Data > BF1_BR1 should be moved to metaBEAT_Bioinformatic_Processing > BF1-BR1 > raw_reads. To run metaBEAT, you will have to install Docker on your computer. Docker is compatible with all major operating systems, but see the Docker documentation for details. On Ubuntu, installing Docker should be as easy as: sudo apt-get install docker.io Once Docker is installed, you can enter the environment by typing: sudo docker run -i -t --net=host --name metaBEAT -v $(pwd):/home/working chrishah/metabeat /bin/bash This will download the metaBEAT image (if not yet present on your computer) and enter the 'container', i.e. the self contained environment (NB: sudo may be necessary in some cases). With the above command, the container's directory /home/working will be mounted to your current working directory (as instructed by $(pwd)). In other words, anything you do in the container's /home/working directory will be synced with your current working directory on your local machine. Please process sequence data belonging to each primer set individually (i.e. do not process sequence data belonging to different primer sets together). An example of expected outputs can be seen in the Jupyter Notebook for the BF1/BR1 primer set from the 2019-11-11 sequencing run. 4_Illinois_Invert_Reference_Database.zip This zipped folder contains all files that were used to generate the custom COI and 16S reference databases for invertebrates that occur in Illinois, U.S. You will need to have metaBEAT installed (see above) before you try to run any Jupyter Notebooks (.ipynb files). 5_ecoPCR.zip This zipped folder contains all files used to perform ecoPCR for each primer set evaluated for microfluidic eDNA metabarcoding. You will need to install ecoPCR before running any shell scripts. 6_Tidied_Data.zip This zipped folder contains the taxonomically assigned data for both sequencing runs produced by metaBEAT and Anacapa. These were copied over from the folders 2_Anacapa_Bioinformatic_Processing and 3_metaBEAT_Bioinformatic_Processing and rearranged into a more logical order. These files are used as the input for data analysis using R. 7_Data_Analysis.zip This zipped folder contains all scripts and metadata required to summarise and statistically analyse data in R. Please contact Dr Lynsey Harper (lynsey.harper2@gmail.com) or Dr Mark Davis (davis63@illinois.edu) if you encounter any issues!

We are grateful for the Strategic Environmental Research and Development Program (SERDP) for funding this research.