DOI: 10.5281/zenodo.17951395
title: Datasets associated with paper titled "Swabbing as a Non-lethal DNA Collection Method for Earthworm Barcoding: Performance and Citizen Science Perspectives"

description: Lethal sampling followed by destructive tissue subsampling is employed routinely in earthworm DNA barcoding studies. However, this approach may be ecologically disadvantageous and risks public disengagement with biodiversity and barcoding research due to negative emotional associations, particularly in Citizen Science (CS) projects. The study describes the performance of swab sampling as a non-lethal and emotionally-adaptive alternative to tissue sampling in earthworm barcoding. Three DNA collection methods, namely swab sampling with silica-based DNA extraction (Swab-QIAGEN), tissue sampling with silica-based DNA extraction (Tissue-QIAGEN), and tissue sampling with resin-based DNA extraction (Tissue-Chelex) were compared for generating cytochrome c oxidase I (COI) barcodes from 40 adult earthworms. All methods showed comparably high PCR amplification success rates (Tissue-Chelex=100%; Tissue-QIAGEN=100%; Swab-QIAGEN=97.5%), expect for one recalcitrant Swab-QIAGEN sample. Considering the pool of successfully amplified samples, the sequencing success rate for the Tissue-Chelex, Tissue-QIAGEN, and Swab-QIAGEN methods was 82.5% (33/40), 87.50% (35/40), and 66.7% (26/39), respectively—with 25 of 119 barcodes flagged as methodological failures due to unusable contigs, stop codons/indels, or contamination. Although swabs showed lower success rates overall, they produced the greatest proportion of reference-quality barcodes among functional sequences (80.8%; 21/26), indicating their strong potential for DNA collection compared to Tissue-Chelex (75.8%; 25/33) and Tissue-QIAGEN (68.6%; 24/35). All intra-specimen barcodes, excluding contaminations, received consistent homology-based taxonomic assignments with average similarity percentage above 99%, corresponding to a 90.4% morphospecies-genetic data concordance rate. The average intra-specimen pairwise sequence percentage identity was above 99% (<0.1% average sequencing error) across all methods, and no method-specific clustering was observed in the phylogenetic tree. Overall, the results indicated a high degree of interoperability and agreement between swab and tissue sampling methods for recovering the same or nearly-identical haplotypes with consistent taxonomic identities from each specimen. However, the swabbing methodology requires standardization before it can be confidently utilized in CS-based earthworm DNA barcoding projects.
description: This repository contains COI barcode sequence data generated from earthworm specimens using three DNA collection methods and bidirectional Sanger sequencing with Folmer primers, underpinning the results and discussions in the aforementioned manuscript.

Files provided (two Fasta files and one Excel file):

SpecimenID_Morphospecies.xlsx

Lists the specimen IDs used for each barcode and the corresponding morphospecies assigned to each specimen. 

primary_dataset.fasta

Contains all functional COI barcode sequences retained after quality filtering and used in the main analyses of the study.

Sequences represent up to three barcodes per specimen (one per DNA collection method, where successful), vary in sequence quality (e.g., length, ambiguity rate, contiguous high-quality regions) and, do not contain stop codons or frameshifts in the translated COI reading frame.

global_dataset.fasta

Contains all sequences from the primary dataset plus additional COI sequences with stop codons that were excluded from the primary dataset but retained for exploratory and phylogenetic analyses. The results of analyses using the global dataset are disclosed in the supporting information provided along with the manuscript.Sequences with stop codons are included for transparency and reproducibility and should not be treated as reference-quality barcodes.

FASTA header format>SpecimenID_Genus_species

SpecimenID is a unique specimen identifier; Genus_species reflects the morphological identification at the time of analysis.

Intended use

The primary dataset underpins the core methodological comparisons presented in the study.The global dataset provides a complete record of all generated sequences.