DOI: 10.5061/dryad.8w9ghx3wv
title: Data from: Optimization of wetland environmental DNA metabarcoding protocols for Great Lakes region herpetofauna

description: Many species of reptiles and amphibians (herpetofauna) rely on wetlands
 that are being degraded and lost at a high rate. Characterization of
 herpetofauna diversity in different wetland types may help guide
 conservation strategies. However, traditional survey methods often involve
 sampling within small temporal windows and gear deployed may be
 taxonomically biased, thus, they may fail to accurately characterize
 species presence/absence and diversity. In contrast, environmental (e)DNA
 metabarcoding has been shown to effectively survey entire aquatic
 communities and can provide a useful complement to traditional surveys.
 The objective of this study was to design and optimize eDNA sampling and
 laboratory protocols for wetland herpetofauna. Protocols evaluated
 included different water sampling approaches (point versus transect
 sampling), seasonality of sampling, and choice of metabarcoding marker
 (mitochondrial 12S versus 16S rDNA). Samples collected from 10 sites
 across southern Michigan detected 17 amphibian and five reptile species,
 including four species of conservation concern (Ambystoma texanum, Clemmys
 guttata, Rana palustris, and Sternotherus odoratus). We observed no
 difference in the number of species detected between point and transect
 samples (p = 0.70), but point sampling required less time (p = 0.03) and
 allowed significantly larger volumes of water to be filtered (p =
 1.13e-5). No difference in species richness was observed between the 12S
 and 16S mitochondrial DNA markers (p = 0.96). However, a greater number of
 taxa were identifiable at the species level when using the 16S locus.
 There was also a significant difference in the number of species detected
 between early and late summer sampling periods (more species detected in
 the earlier period; p = 6.31e-6), and some species were only found in the
 early or late sampling period. We recommend sampling during multiple
 periods to fully characterize species composition, the use of point
 sampling, and the 16S mtDNA marker for herpetofauna eDNA metabarcoding
 studies.
description: # Data from: Optimization of wetland environmental DNA metabarcoding
 protocols for Great Lakes region herpetofauna
 [https://doi.org/10.5061/dryad.8w9ghx3wv](https://doi.org/10.5061/dryad.8w9ghx3wv) This repository contains analysis scripts and input data files associated with the eDNA metabarcoding of herpetofauna communities in Michigan wetlands and subsequent analyses for “Optimization of wetland environmental DNA metabarcoding protocols for Great Lakes region herpetofauna”.  ## Description of the data and file structure Alignment and taxonomy files for 279 species (12S) and 305 species (16S) are in the `alignment_taxonomy.zip` file.  * `Vertebrate_12S_align_072821_NoPrimers.fas` and `Vertebrate_16S_align_111722_NoPrimers.fas` contain sequence alignments for species with accession numbers (if available) and scientific names. If produced by our lab, there is no accession number, but a four-letter code (first two letters of genus and specific epithet) and the sample number.  * `Vertebrate_12S_rDNA_taxonomy_072821.txt` and `Vertebrate_16S_rDNA_taxonomy_111722.txt` contain accession or lab-produced numbers and the full taxonomic classifications for each species.  Cleaned community matrices with target herpetofauna species as `.csv` files are in the `community_matrices.zip` file. Included scripts to create community matrices are described below in the “Code/Software” section.  * `clean_community_matrix_12S_y1.csv`/`clean_community_matrix_16S_y1.csv`- a data file containing read counts for herpetofauna species detected in samples for the 12S or 16S markers. * Each file contains the following columns: 1. sample- sample name with the following structure: * Example: 16S_JC2_T5.16S * 16S, the marker * C2, Jackson county site 2 * T, transect sample  * 5, fifth transect sample * 16S, marker 2. OTU (operational taxonomic unit) read counts (e.g. Rana_clamitans) for each sample in the data set Files used for the Mothur (Schloss, 2009) analysis are included in the `mothur_files.zip` file. * `MSU_12S_eDNA_oligos_file.txt`/`MSU_16S_eDNA_oligos_file.txt`- a text file containing barcodes required for each marker analysis. These files contain a sample column in the following structure:  1. 12S/16S- column labeled with the marker * Example: 2BR1_T1 * 2, the second sampling period * BR1, the first Barry county site * T, transect sample  * 1, first transect sample * EN or Neg are extraction or PCR negatives Output files produced from sequencing can be found in the `post_seq_files.zip` file. This directory contains the following four gzipped fastq files: * `OR_2021_Herp_P_Lib_S1_L001_R1_001.fastq.gz` and `OR_2021_Herp_P_Lib_S1_L001_R2_001.fastq.gz`- One file for read one and one file for read two. * `OR_2021_Herp_P_Lib_S1_L001_I2_001.fastq.gz` and  `OR_2021_Herp_P_Lib_S1_L001_I1_001.fastq.gz`- One file for forward indices and one file for reverse indices. Data required for analyses of our data set are included in the `analysis_data.zip` file. This directory contains 10 `.csv` files. * The file `full_herp_data.csv` is the primary file to be used for most analyses. It contains the following columns: 1. sample- sample name with the following structure: * Example: 2BR1_T1 * 2, the second sampling period * BR1, the first Barry county site * T, transect sample  * 1, first transect sample 2. type- sampling method, point or transect  3. vol-  amount of water collected for a sample             4. season-  sampling period (Period 1 or Period 2/spring or summer) 5. wetland-  wetland type sampled (m = marsh, vp = vernal pool, f = fen)    6. duration-  the amount of time in second it took to filter the record volume of water for a sample                             7. no_spp-  the number of species detected in that sample * The file `specpool_env.csv` is a truncated version of the file above, including only the sample, sample type, sampling period, and wetland type.  * The following files have column names with a genus and specific epithet included (Example: Rana_clamitans): `finalspec.csv`, `forshan24.csv`, `fullspec_herp.csv` and `specpoolexp.csv`.  * The files `mcnumbers12S.csv` and `mcnumbers16S.csv` contain expected and observed read proportions and species names assessed for mock communities and marker correlation analyses.  * The file `nmds_new.csv` contains the following columns: 1. sample- sample name with the following structure: * Example: 2BR1_T1 * 2, the second sampling period * BR1, the first Barry county site * T, transect sample  * 1, first transect sample 2. period- sampling period (Period 1 or Period 2/spring or summer) 3. county- name of sampling site county 4. sampletype- type of sampling method (point or transect) 5. OTU (operational taxonomic unit) relative read counts (e.g. Rana_clamitans) for each sample in the data set * The file `shannonenv.csv` is a list of county names for Shannon diversity analyses. * Site abbreviations for all above files are as follows: * CLI-  Clinton county * BR1-  Barry county, site 1 * BR2-  Barry county, site 2 * KZ1-  Kalamazoo county, site 1 * KZ2-  Kalamazoo county, site 2 * LIV-  Livingston county * OAK-  Oakland county  * JC1-  Jackson county, site 1 * JC2-  Jackson county, site 2 * HIL-  Hillsdale county ## Code/Software Bioinformatic processing scripts can be found in the `mothur_scripts`/ subdirectory of `data_processing.zip`. This includes five scripts sorted by mitochondrial marker (12S or 16S). * `Herp_12S_batch_mothur_script_OMR.sh`/`Herp_16S_batch_mothur_script_OMR.sh`- a script of commands that run through the Mothur pipeline on a high-performance computing cluster. * `submission_12S_herp.sh`/`submission_16S_herp.sh`- a script for submission to call the executable batch Mothur script described above. * `MakeContigs_12S_TEMPLATE.sh`- a template script for the `make.contigs` step in the Mothur pipeline.  * Required oligos files are in the `mothur_files/` directory described above. Additionally, R code to create community matrices can be found in the `otu2cm/` subdirectory of `data_processing.zip` file. This directory contains the following two R scripts. * `step1_OTU_classification_summary_herps.R`- A script to evaluate the OTU classifications made by the Mothur pipeline and write processed taxonomic groups to an out file. * `step2_OTU2_condensed_community_matrix_JR_FAST_herps.R`- A script to assess OTU classifications from Mothur and create community matrices. R code for analyses and visualization associated with the manuscript are in the `analyses/` directory of the `analyses.zip` file.  * `Analyses_Plots_HerpeDNA.R` - Analyses included in this script: ANOVAs, PERMANOVAs, NMDS plots, boxplots, paired t-tests, mock community plots, and correlation tests - All required data to reproduce analyses and plots in this script are in the `analysis_data/` directory described above.