One Size Does Not Fit All: Tuning Edna Protocols For High-And Low-Turbidity Water Sampling Environmental DNA

Findings from eDNA metabarcoding are strongly influenced by experimental approach, yet the effect of pre-PCR sample processing on taxon detection and estimates of biodiversity across different water types is still poorly resolved. To fill this data gap, we investigated the impact of sampling effort, extraction method, and filter pore size on DNA yield, PCR inhibition, and 16S rDNA metabarcoding results for fishes in water samples collected from inshore turbid- and offshore clear-water environments. The turbid-water samples had high concentrations of suspended organic and/or inorganic material and yielded ~3.2× more DNA and exhibited high levels of PCR inhibition compared with the low-turbidity, clear-water samples. Importantly, there were no striking differences in the results of our metabarcoding experiments based on extraction method or filter pore size. While a small number of unique species of relatively low read count were detected in all turbid-water treatments, most species were consistently detected across samples. Results for the clear-water samples were strikingly different, with low DNA yield, high levels of variation across replicates, and a high number of non-overlapping species across treatments. These findings indicate a patchy distribution of eDNA in offshore environments, which means higher volumes of water (≥ 2 L per replicate) must be filtered in habitats where target DNA is likely to be sparse. In semi-closed systems such as estuaries, higher concentrations of target DNA are expected, and we found that either a 1.0 or 3.0 µm filter pore size was sufficient to capture standing diversity, while decreasing the risk of clogging. For economical DNA extraction and inhibitor removal, we recommend a combination of Omega Bio-tek E.Z.N.A Tissue DNA kit followed by a PCR inhibitor removal step using the Zymo Kit. Finally, we emphasize that pilot studies should be undertaken whenever sampling in a new environment to identify which protocol is most appropriate. Raw demultiplexed fastq files have been uploaded to GenBank's Sequence Read Archive (SRA; BioProject ID: PRJNA742717) and the associated metadata can be found at the Genomic Observatories MetaDatabase (GEOME; https://geome-db.org/; Deck et al., 2017). ASV files with read counts and corresponding sequences can be found on Data Dryad (https://doi.org/10.5061/dryad.ghx3ffbp6)

环境DNA元条形码（eDNA metabarcoding）的研究结果受实验方案的影响极大，但目前针对不同水体类型，PCR前样本处理对类群检测及生物多样性评估的影响仍未得到充分阐明。为填补这一数据空白，我们针对从近岸浑水环境与远洋清水环境采集的水样，研究了采样投入、DNA提取方法及滤膜孔径对鱼类DNA得率、PCR抑制效应及16S核糖体DNA（16S rDNA）元条形码测序结果的影响。结果显示，相较于低浊度清水样本，浑水样本中悬浮有机/无机颗粒物浓度更高，DNA得率约为其3.2倍，且表现出更强的PCR抑制水平。值得注意的是，本研究的元条形码实验结果未因DNA提取方法或滤膜孔径出现显著差异。尽管在所有浑水处理组中仅检测到少量读长计数较低的特有物种，但大多数物种在各样本中均可稳定检出。而清水样本的结果则截然不同：DNA得率较低，重复样本间变异程度高，且各处理组间非重叠物种数量较多。上述结果表明，远洋环境中eDNA呈斑块状分布，这意味着在靶标DNA较为稀缺的生境中，需过滤更大体积的水样（每个重复≥2升）。在半封闭系统（如河口）中，靶标DNA浓度通常更高；本研究发现，采用1.0 μm或3.0 μm的滤膜孔径即可捕获现存生物多样性，同时降低滤膜堵塞风险。出于经济高效的DNA提取与抑制物去除需求，我们建议采用Omega Bio-tek公司的E.Z.N.A组织DNA试剂盒，后续搭配Zymo试剂盒完成PCR抑制物去除步骤。最后，我们强调，在全新环境中开展采样前，应先行开展预实验以确定最适配的实验方案。原始解复用fastq文件已上传至GenBank序列读取档案库（Sequence Read Archive, SRA；生物项目编号：PRJNA742717），相关元数据可在基因组观测元数据库（Genomic Observatories MetaDatabase, GEOME；https://geome-db.org/；Deck等，2017）中获取。包含读长计数与对应序列的扩增子序列变异（ASV）文件可在Data Dryad数据库获取（https://doi.org/10.5061/dryad.ghx3ffbp6）。