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.