Advancing responsible genomic Analyses of ancient mollusc shells. Methods ancient mollusc shell genomics

The analysis of the DNA entrapped in ancient shell specimens of molluscs has the potential to shed light on the evolution and ecology of this very diverse phylum. Ancient genomics using high-throughput DNA sequencing (HTS) technologies could help reconstruct the responses of molluscs to past climate change, pollution events, aquaculture practices and extinction events at unprecedented temporal resolutions. Such studies are however still lacking. This is partly due to our limited knowledge of the DNA preservation in calcium carbonate shells and the need for optimized methods for genomic data generation from ancient shells. The latter is crucial for efficient and responsible research conduct, as ancient DNA analyses partially destroy specimens that belong to our heritage. Here, we applied HTS to 27 mollusc shells dated to 111-7,000 years Before Present in order to improve ancient shell genomic analyses. In particular, we investigated the potential impact of X-ray radiation on ancient shell DNA during micro computed tomography (micro-CT) scanning for the preservation of morphological information. We then compared the performance of commonly used silica-based DNA extraction methods and predigestion treatments, and finally contrasted DNA recovery from the different organic (periostracum) and carbonated (aragonite, calcite) layers that compose mollusc shells. We first show that micro-CT scanning allows the conservation of their 3D shape without any detectable impact on their DNA. We then suggest that DNA is protected from degradation and contamination in preservation niches within ancient shells, so that DNA recovery was improved by double-digestion and predigestion bleach wash of shell powder prior to silica-based DNA extraction. The periostracum, as well as the calcite and aragonite layers, constitute valuable DNA reservoirs, with aragonite appearing as the best substrate for genomic analyses. Our results allowed us to propose recommendations that we anticipate will be helpful for future efficient and responsible genomic analyses of ancient mollusc shells. Our work also contributes to the understanding of long-term molecular preservation in biominerals.