Statistical test results.

The analysis of the DNA entrapped in ancient shells of molluscs has the potential to shed light on the evolution and ecology of this very diverse phylum. Ancient genomics could help reconstruct the responses of molluscs to past climate change, pollution, and human subsistence practices at unprecedented temporal resolutions. Applications are however still in their infancy, partly due to our limited knowledge of DNA preservation in calcium carbonate shells and the need for optimized methods for responsible genomic data generation. To improve ancient shell genomic analyses, we applied high-throughput DNA sequencing to 27 Mytilus mussel shells dated to ~111–6500 years Before Present, and investigated the impact, on DNA recovery, of shell imaging, DNA extraction protocols and shell sub-sampling strategies. First, we detected no quantitative or qualitative deleterious effect of micro-computed tomography for recording shell 3D morphological information prior to sub-sampling. Then, we showed that double-digestion and bleach treatment of shell powder prior to silica-based DNA extraction improves shell DNA recovery, also suggesting that DNA is protected in preservation niches within ancient shells. Finally, all layers that compose Mytilus shells, i.e., the nacreous (aragonite) and prismatic (calcite) carbonate layers, with or without the outer organic layer (periostracum) proved to be valuable DNA reservoirs, with aragonite appearing as the best substrate for genomic analyses. Our work contributes to the understanding of long-term molecular preservation in biominerals and we anticipate that resulting recommendations will be helpful for future efficient and responsible genomic analyses of ancient mollusc shells.对保存在古代软体动物贝壳中的DNA开展分析，有望为这一多样性极高的门类的演化与生态研究提供全新洞见。古代基因组学研究可帮助以前所未有的时间分辨率，重建软体动物对过往气候变化、污染以及人类生存活动的响应模式。不过该领域的应用仍处于起步阶段，部分原因在于我们对碳酸钙贝壳中DNA保存机制的认知较为有限，且亟需开发优化方法以实现合规的基因组数据生成。为优化古代贝壳基因组分析流程，本研究对27件年代介于距今约111至6500年的贻贝（Mytilus）贝壳开展高通量DNA测序，并探究了贝壳成像、DNA提取方案以及贝壳亚采样策略对DNA回收效率的影响。首先，我们发现，在亚采样前利用显微计算机断层扫描（micro-computed tomography）记录贝壳三维形态信息，不会对DNA产生定量或定性的有害影响。其次，本研究证实，在基于硅胶基质的DNA提取流程前，对贝壳粉末进行双酶消解与漂白处理，可提升贝壳DNA的回收效率，这一结果也表明，古代贝壳内部的保存微环境可对DNA起到保护作用。最后，研究证实，贻贝贝壳的所有组成层——无论是带有还是不带外层有机壳皮（periostracum）的珍珠质（文石（aragonite））层与棱柱质（方解石（calcite））碳酸盐层——均为极具价值的DNA储存库，其中文石层被证明是基因组分析的最优底物。本研究有助于深化对生物矿物中长期分子保存机制的认知，我们期待本次研究提出的建议，能够为未来古代软体动物贝壳的高效且合规的基因组分析提供助力。