The CODEX Approach: High-Throughput Sequencing of the Cox-1 Barcode Fragment in Neogastropods (Mollusca, Gastropoda).

Abstract

DNA barcoding traditionally relies on Sanger sequencing but faces limitations with degraded samples. High-throughput sequencing (HTS) offers a cost-effective alternative, enabling rapid barcode generation for extensive datasets. The advantage of HTS is its ability to employ multiplexing strategies, allowing thousands of samples to be processed simultaneously in a single sequencing run. This study presents the CODEX approach, a double-indexed HTS method designed to sequence overlapping cox-1 barcode fragments, suitable for samples with degraded DNA. The approach was applied to neogastropods, a diverse lineage of marine molluscs, using specimens (both recently collected and relatively older) from the Muséum national d'Histoire naturelle (MNHN) collections. The pipeline was used to process 15,076 samples, yielding 10,905 cleaned and assembled sequences, achieving a success rate of 72.33%. The CODEX method demonstrated advantages over Sanger sequencing by enabling the recovery of barcodes from samples previously deemed unsuitable, with significantly reduced costs (€0.5 per sequence vs. €4.5). Notably, DNA quality and sequencing success were strongly correlated with collection date, emphasising the impact of preservation methods and storage conditions. Sequencing success rates varied among families but were not correlated with phylogenetic relationships or specimen size, indicating the robustness of the primers designed for neogastropods. This study highlights the efficiency of the CODEX approach for large-scale DNA barcoding projects, especially when handling degraded samples. The CODEX pipeline and associated resources are publicly accessible, offering a scalable solution for molecular systematics and beyond.