DOI: 10.5061/dryad.g1jwstr28
title: Data and code from: Establishing DNA-based strategies for soil biodiversity assessment: Insights from carabid beetles

description: Molecular-based methods offer valuable opportunities for assessing soil
 biodiversity in different ecosystems. However, their reliability and
 large-scale applicability depend on developing, optimizing protocols, and
 establishing high-quality, curated local reference databases. This study
 aimed to evaluate key steps in the soil macroinvertebrate metabarcoding
 workflow, including the sample decontamination process and the efficiency
 of taxa recovery. Specifically, we sought to: (1) determine the impact of
 sample decontamination, (2) validate species-level recovery efficiency of
 the metabarcoding pipeline spiked with a curated mock community of
 morphologically identified and barcoded carabid beetles, and (3) compare
 traditional morphological identification and metabarcoding for specimens’
 taxonomic assignment and recovery. Our results showed that the commonly
 used decontamination process did not significantly impact OTU richness,
 suggesting it is not essential for this fauna. Compared to morphology,
 metabarcoding provided a more comprehensive taxonomic overview at
 higher-level taxa. However, validation with the mock community revealed
 discrepancies in species-level recovery, underscoring that its accuracy is
 highly contingent on the quality of the reference database. DNA
 metabarcoding is a currently used and promising technique for
 macroinvertebrate assessment regarding time, efficiency, and costs, yet
 reaching greater depth in taxonomic resolution. Yet, its species-level
 accuracy remains dependent on comprehensive and well-curated barcode
 reference databases. We recommend an integrative approach, combining
 molecular data with targeted validation, for the most robust outcomes. For
 this reason, we recommend the use of integrative methodologies for robust
 and rapid biodiversity assessments. We found that the common
 decontamination step is not crucial for soil macrofauna metabarcoding
 accuracy. Consequently, its removal streamlines sample
 processing. DNA metabarcoding revolutionizes soil biodiversity
 assessments by offering unparalleled taxonomic resolution and efficiency
 compared to traditional methods. Our study shows that the common
 decontamination step is unnecessary, streamlining workflows without
 compromising accuracy. However, the reliability of these molecular methods
 hinges on the development of curated local reference databases,
 underscoring the importance of integrative approaches for robust
 biodiversity monitoring. 
description: Sample
 Collection and Processing This study, conducted under
 the CULTIVAR project in Idanha-a-Nova, Portugal, monitored soil macrofauna
 across 24 plots, each containing nine pitfall traps. A total of 216 traps
 were set up from late November to early December 2022, remaining in the
 field for 13 to 17 days. Traps were filled with ethylene glycol as a
 preservative and covered with lids to prevent dilution by rain. Collected
 specimens were preserved in 96 % ethanol until further analysis. Under
 low-power microscopy, organisms were counted and identified, per sample,
 to the lowest possible taxonomic level using morphology-based keys and
 literature. These community samples were stored at 96 % ethanol.
 Establishing a COI Barcode Reference Database Carabidae specimens were identified to the species level using morphological traits. To crete a local barcode reference base, one to two identified specimens were bleached, washed, and DNA was extracted using a Qiagen DNeasy® kit. For smaller specimens, a non-destructive method was applied, while larger specimens required maceration of appendages. PCR amplification of a 710 bp COI sequence utilized Folmer primers. Amplified products were verified via agarose gel electrophoresis, purified, and sequenced through the Sanger method. Sequences were analyzed against the BOLD and NCBI databases, allowing taxonomic assignments. A mock community of 31 Carabid species was created to validate metabarcoding methods, such as the recovery efficiency. <strong>Bulk Sample Preparation and Metabarcoding</strong> Half of the community samples went through a decontamination process using sodium hypochlorite. All community samples were homogenized into fine powder using a Bullet Blender. DNA was extracted with an E.Z.N.A.® Tissue DNA Kit. Carabid beetles were excluded as they were part of a mock community. PCR amplification targeted a 418 bp COI fragment using primers BF3 and BR2. A two-step PCR process incorporated Illumina adapters and identification indices. Final libraries were quantified, pooled, and sequenced using the Illumina NovaSeq platform. Controls ensured no contamination during amplification. Bioinformatics Pipeline The pipeline employed OBITools 4 for sequence processing, VSEARCH for denoising, and LULU for filtering pseudogenes. Paired-end reads were merged, primer sequences removed, and OTUs grouped by 99 % similarity. Taxonomic assignments were made using BOLD and NCBI databases. The script is available as a .qmd document.  Comparative Analysis and Statistical Validation Morphological and molecular identification methods were compared at the order and species levels. Statistical analyses were performed using R, (script available as a .qmd document). A barcode gap of Carabid species of 7 % was establishid to define species delimitation. This was accomplished by joining our Carabid sequences with more than 600 sequences retrieved from NCBI (sequence accession number available in the dataset), using MEGA and genetic distances calculated via the K80 model. This allowed the creation of a Venn diagram for comparing species and genus recovery efiency of barcoding and metabarcoding compared to morphology.
description: # Data and code from: Establishing DNA-based strategies for soil
 biodiversity assessment: Insights from carabid beetles
 [https://doi.org/10.5061/dryad.g1jwstr28](https://doi.org/10.5061/dryad.g1jwstr28) ## Description of the data and file structure This repository contains the data and the scripts from a study conducted as part of the CULTIVAR project in Idanha-a-Nova, Portugal. The research focused on monitoring soil macrofauna diversity through a combination of traditional morphological identification and advanced molecular techniques, such as barcoding and metabarcoding. ## Study Overview * **Sample Collection**:\ Soil macrofauna were collected from 24 plots using 216 pitfall traps set up from late November to early December 2022. Specimens were preserved in 96 % ethanol and sorted under low-power microscopy. * **COI Barcode Reference Database**:\ Carabidae specimens were identified morphologically and DNA barcoded using Folmer primers to establish a local COI reference database. Sequences were matched to BOLD and NCBI for taxonomic assignments, and a mock community of 31 Carabid species was assembled for validation. * **Metabarcoding Workflow**:\ Bulk community samples were decontaminated, homogenized, and DNA-extracted. A 418 bp COI fragment was amplified using a two-step PCR process and sequenced via Illumina NovaSeq. The bioinformatics pipeline used is provided in this repository as "BioinformaticPipeline.qmd". * **Comparative Analysis**:\ Decontamination efficiency and the comparison of morphological identifications, barcoding and metabarcoding results using R-based statistical analyses are here available as "DataAnalysis.qmd". A 7 % barcode gap was employed for species delimitation, integrating project sequences with NCBI data using Assemble Species by Automatic Partitioning (ASAP), ASAP Web results are also availale. ## Files and variables ### BioinformaticPipeline.qmd Quarto document used to process the raw metabarcoding sequences. It requires `OBITools`v4.3.0 , `ROBIFastread` v0.1.0 , `VSEARCH` v1.2.13, `LULU`v0.1.0 and `boldigger` v2.2.2 and the `ngs_fiter.py` python script. OBITools 4 was used for the sequence processing, coupled with VSEARCH and LULU (Frøslev et al., 2017) for denoising. Paired-end reads were aligned and merged (sequences that failed to overlap were removed). With `ngs_fiter.py` the reads were assigned to samples, and primer sequences were removed. Merged reads were collapsed into haplotypes. Singletons are then removed using . Then, each remaining haplotype was combined into a single file and once more dereplicated. PCR, sequencing error and chimeric sequences, are removed through the pipeline. The remaining sequences were then grouped by a 99 % similarity criterion to define operational taxonomic units (OTUs). Putative NUMTs (nuclear copies of the mitochondrial COI) were then identified and removed using LULU. All remaining OTUs were identified by comparing the representative OTUs of each cluster against online databases through Boldigger. Only sequences of targeted taxa went through downstream analysis. ### DataAnalysis_Revised_10_10_25.qmd Script contains all statistical analyses and data visualization used for the associated publication. These were performed in R v2023.06.0 + 421 using packages' `iNEXT`, v3.0.0. , for diversity estimates, `ggplot2`, v3.4.2 and `ggpubr` v0.6.0, for data visualization, and `ggVennDiagram` v1.12, for the creation of a Venn diagram. ### File: Data.zip **Description:** ZIP file containg all the data separated by type of analysis. Missing data conventions: `NA` = not applicable. This file contains three folders: #### **1.Morpho** Contains one excel file ##### `MorphoAnalysis.xlsx` Description: file with the abundance of morphologically sorted organisms. Samples were sorted under a low-power microscope, where organisms were counted and identified to the lowest possible taxonomic level based on morphological traits through dichotomous keys. | Variable name | Description | **Units / Categories / Notes** | | ---------------- | ---------------------------------------------- | ------------------------------ | | sample | Unique pitfall sample label. | Text | | Agroecosystem | Agroecosystem classification of the plot. | Categorical | | TotalAbundance | Total number of individuals in that trap | Count | | Araneae | Number of individuals from that group per trap | Count | | Archaeognatha | Number of individuals from that group per trap | Count | | Blattodea | Number of individuals from that group per trap | Count | | Dermaptera | Number of individuals from that group per trap | Count | | Gastropoda | Number of individuals from that group per trap | Count | | Hemiptera | Number of individuals from that group per trap | Count | | Isopoda | Number of individuals from that group per trap | Count | | Opiliones | Number of individuals from that group per trap | Count | | Orthoptera | Number of individuals from that group per trap | Count | | Pseudoscorpiones | Number of individuals from that group per trap | Count | | Coleoptera | Number of individuals from that group per trap | Count | | Hymenoptera | Number of individuals from that group per trap | Count | | Myriapoda | Number of individuals from that group per trap | Count | #### **2.OTU** Contains 3 files with the results and resources for the metabarcoding/ OTU-based molecular analyses. ##### `BOLDResults_curated.xlsx` Description: Excel file containing 3 sheets: ##### - "BOLDigger hit" First BOLDigger hit for each OTU, with final "keep?" decision, with `NAs` added as Not Aplicable | Variable name | Description | | ------------------------------------------------- | --------------------------------------------------------------- | | ID | OTU identifier | | Phylum / Class / Order / Family / Genus / Species | Taxonomic ranks returned by BOLD | | Similarity | % identity to BOLD record | | Status | BOLDigger assignment status | | Flags | BOLDigger flags | | Keep? | YES / NO flag: whether OTU was retained for downstream analyses | --- ##### - "RAW_BOLDigger hit" Raw first-hit assignments directly exported from BOLDigger, before manual curation. NAs were not added and "Keep?" columns does not exist. | Variable name | Description | | ------------------------------------------------- | --------------------------------------------------------------- | | ID | OTU identifier | | Phylum / Class / Order / Family / Genus / Species | Taxonomic ranks returned by BOLD | | Similarity | % identity to BOLD record | | Status | BOLDigger assignment status | | Flags | BOLDigger flags | | Keep? | YES / NO flag: whether OTU was retained for downstream analyses | --- ##### - "Annotation_07-12-2023" Full raw annotation table exported from BOLD, with the first 20 hits for each OTU. | Variable name | Description | | -------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | You searched for | Query sequence submitted | | Phylum / Class / Order / Family / Genus / Species / Subspecies | Taxonomic ranks | | Similarity | % similarity | | Status | BOLD status | | Process ID | Unique identifiers for the DNA sequencing process. | | Record ID | Unique identifiers for the resulting specimen record. | | BOLD BIN | The [Barcode Index Number](https://www.google.com/search?q=Barcode+Index+Number\&rlz=1C5CHFA_enPT952PT952\&oq=You+searched+for%09Phylum%09Class%09Order%09Family%09Genus%09Species%09Subspecies%09Similarity%09Status%09Process+ID%09Record+ID%09BOLD+BIN%09Sex%09Life+stage%09Country%09Identifier%09Identification+Method%09Institution+storing%09Specimen+page+url\&gs_lcrp=EgZjaHJvbWUyBggAEEUYOdIBBzYyMWowajeoAgCwAgA\&sourceid=chrome\&ie=UTF-8\&mstk=AUtExfBAa0BAZkOzY3WH8K5dBLDaS6_DF1vOjpoF3gL84zQFTyE5XP-QvyPcc0GBNlw0ZxiJG8zcJgv42HdkoUXaMOCnLJtwrdFe3vla66trN2LKpbo9XHGbA0mwMAD8If4Z45OjLzGPIUd6S9s8oGxgCyjEYgwU4iu7xBh-6kizdLg-Y7lzc9tJUajYa9JnQnpNDfsJWw03mFkn74Cg9fPHBmSt5tZuU9JOiRoA27AD-1FuO8Eb1HRevUq2gOeVnet7_P_V9YO768DUubJh1VCe9eGD\&csui=3\&ved=2ahUKEwi44e2_y9uQAxUPOPsDHWhvHRMQgK4QegQIAxAD) associated with a specific DNA barcode sequence within the BOLD database | | Sex | sex of voucher (if known) | | Life stage | life stage (if known) | | Country | The geographical location where the specimen was collected | | Identifier | The name of the researcher who submitted or identified the specimen. | | Identification Method | Method used for ID | | Specimen page url | Direct web link to the individual specimen's record page | ##### `otutable_curated.tsv` Raw file with the number of OTU reads per sample. ##### `otutable_keptOTUs.xlsx` Data analysis output. File with the number of filtered OTU reads per sample. Only the retained OTUs: crossed information from otutable_curated with the first BOLDigger hit for each OTU, with final "keep?" decision "Yes". ##### `sample.info.tsv` / `sample.info.xlsx` These files describe each sample included in the metabarcoding dataset.\ They contain the following variables: | **Variable** | **Description** | **Units / Categories / Notes** | | ---------------------- | -------------------------------------------------------------------------------------------------------------------------- | ------------------------------------------------------------------------------ | | `Sample` | Metabarcoding sample identifier corresponding to each pitfall trap. | Text | | `id` | Internal identifier. | Alphanumeric | | `i7 (revcom) - SIF` | Reverse complement of the i7 index used for library preparation. | Nucleotide sequence | | `i5 (revcom) - SIF` | Reverse complement of the i5 index used for library preparation. | Nucleotide sequence | | `i7` | i7 index tag used for demultiplexing. | Nucleotide sequence | | `i7 oligo seq (5′–3′)` | Full oligonucleotide sequence for the i7 primer (forward orientation). | Nucleotide sequence | | `i5` | i5 index tag used for demultiplexing. | Nucleotide sequence | | `i5 oligo seq (5′–3′)` | Full oligonucleotide sequence for the i5 primer (forward orientation). | Nucleotide sequence | | `Combo` | Combination of i7 and i5 indices used for multiplexing. | Text | | `index` | Identifier for the specific index combination used in sequencing. | Text | | `sample` | Unique pitfall sample label. | Text | | `code` | Alphanumeric field code linking season of the pitfall sample (T2= Autumn). | Text | | `Transect` | Transect designation within the study plot. | Text | | `decontamination` | Indicates whether the sample underwent decontamination prior to extraction. | Categorical ("decontaminated", "non-decontaminated") | | `SampleType` | Type of sample. | Categorical ("decontaminated", "non-decontaminated", "BPcr", "MOCK", "NegExt") | | `ext.weight` | Weight of material used for DNA extraction. | grams (g) | | `specimens` | Number of individual specimens in the bulk sample. | Count | | `orders` | Number of taxonomic orders represented in the sample according to morphological identification. | Count | | `batch` | Extraction batch identifier, batches of 24. | Integer | | `extneg` | Extraction negative control identifier according to the batch. | Text / `n/a` if not applicable | | `pcrneg` | PCR negative control identifier. | Text / `n/a` if not applicable | | `dna-plate` | Identifier of the plate. | Text | | `dna-well` | Well position within the sequencing plate. | Alphanumeric (e.g., "A01") | | `mock.test` | Indicates whether the sample is part of the mock community test. | Categorical (TRUE / FALSE) | | `diversity` | Classification of taxa detected per sample from morphological analysis; High diversity &gt;7 order, Low diversity &lt;7 orders . | Categorical ("high", "low") | | `concentration` | Proportion of DNA added to the Mock Test. | Numeric | | `Agroecosystem` | Agroecosystem classification of the plot. | Categorical | ##### BioinformaticPipeline **Folder** containing: * folder with the raw data (01.RawData) * each folder within is named by sample (S1 - S235), each containg the MD5.txt file and four .fq.gz files containg the metabarcoding sequences to be used under the bioinformatic pipeline. * `samplesheet.txt` file. * `ngs_filter.py` file #### **3.Barcode** Folder has the resources and results related to the Carabid taxonomic identification. ##### **ASAP_Result_Idanha.pdf** Description: A report summarizing the results of the ASAP (Assemble Species by Automatic Partitioning) analysis, used for species delimitation. ##### **Appendix S2.xlsx** Description: Excel file with the cytochrome oxidase subunit I (COI) gene sequences for all Carabid beetle specimens included in this study. Each entry provides the GenBank accession number, species identification, and corresponding nucleotide sequence. These data form the basis for the barcode gap and species delimitation analyses presented in the main text. | **Variable** | **Description** | | --------------------- | -------------------------------------- | | `Sequence_ID` | Unique identifier for each sequence | | `Identification` | Species identification | | `GenBank Description` | Metadata associated with GenBank entry | | `Sequence` | Nucleotide sequence (COI gene) | ##### **TaxBarMet.xlsx** A detailed table summarizing taxonomic, barcoding and metabarcoding carabid species identification. It contains three sheets: ##### - "All" Description: Sheet with all the species identified in this study. | **Variable** | **Description** | **Units / Categories / Notes** | | --------------- | ------------------------------------------- | ------------------------------------- | | `Phylum` | Taxonomic phylum | Text | | `Class` | Taxonomic class | Text | | `Order` | Taxonomic order | Text | | `Family` | Taxonomic family | Text | | `Genus` | Taxonomic genus | Text | | `Species` | Species name | Text | | `Taxonomy` | Species presence according to taxonomy | Binomial ( 0 = absence; 1 = presence) | | `Barcoding` | Species presence according to barcoding | Binomial ( 0 = absence; 1 = presence) | | `Metabarcoding` | Species presence according to metabarcoding | Binomial ( 0 = absence; 1 = presence) | ##### - "VennGenus" Description: Sheet with the genus identified by each method. | **Variable** | **Description** | **Units / Categories / Notes** | | --------------- | ------------------------------------------- | ------------------------------------- | | `Phylum` | Taxonomic phylum | Text | | `Class` | Taxonomic class | Text | | `Order` | Taxonomic order | Text | | `Family` | Taxonomic family | Text | | `Genus` | Taxonomic genus | Text | | `Taxonomy` | Species presence according to taxonomy | Binomial ( 0 = absence; 1 = presence) | | `Barcoding` | Species presence according to barcoding | Binomial ( 0 = absence; 1 = presence) | | `Metabarcoding` | Species presence according to metabarcoding | Binomial ( 0 = absence; 1 = presence) | ##### - "Venn\\&gt;=93%" Description: This sheet presents the species-level identifications based on the established barcode gap threshold, including only matches with **≥ 93 % sequence identity**. | **Variable** | **Description** | **Units / Categories / Notes** | | --------------- | ------------------------------------------- | ------------------------------------- | | `Phylum` | Taxonomic phylum | Text | | `Class` | Taxonomic class | Text | | `Order` | Taxonomic order | Text | | `Family` | Taxonomic family | Text | | `Genus` | Taxonomic genus | Text | | `Species` | Species name | Text | | `Taxonomy` | Species presence according to taxonomy | Binomial ( 0 = absence; 1 = presence) | | `Barcoding` | Species presence according to barcoding | Binomial ( 0 = absence; 1 = presence) | | `Metabarcoding` | Species presence according to metabarcoding | Binomial ( 0 = absence; 1 = presence) | ##### - "Final_Classification(TableS2)" Information used to build Table S2 from Appendix S1. According to the agreement on the methods used, DNA barcoding or DNA metabarcoding, species were classified the into "Morphospecies," "IOTUS," "MOTUs," and "Not considered," according to the established barcode gap (ID &lt; 93 %), "0" represents absence. Accordingly: Morphospecies: species only identified morphologically. IOTUS (Integrated Operational Taxonomic Units): species where both morphological and molecular data was available. MOTUs (Molecular Operational Taxonomic Units): species identification that relied only on molecular data.