Optimization of soil eDNA methods using samples from Stornes Peninsula, East Antarctica

Understanding the patterns of distribution of Antarctic terrestrial organisms is crucial if we are to mitigate the effects of environmental change. Important questions that remain unanswered include: where are biodiversity hotspots in Antarctica? Are terrestrial communities biologically isolated or well connected? The rapid development of genomic techniques provides new opportunities to address these and related questions. Here we optimize environmental DNA (eDNA) metabarcoding protocols using soil samples from Stornes Peninsula, East Antarctica. DNA sampling, DNA extraction and library preparation methods were compared to search for a low cost- and time-consuming eDNA metabarcoding protocol to assess the diversity of plants and animals using low-biomass soil samples. Our results show that comparatively similar OTU data sets can be retrieved using low volumes of starting material, in combination with time and cost-efficient extraction and library preparation methods. Also, and compared to other regions, low taxa were found. These results are highly valuable for future monitoring of Antarctic terrestrial biota. Soil samples were collected in Stornes Peninsula, East Antarctica (69°24.011'S, 76°05.382'E), in December 2018. Around 50 ml of soil samples from the surface was collected from each corner of a 1 m2. The four soil samples were further processed both individually and as a combined sample. Next, each replicate was DNA extracted with the following methods: (1) PowerSoil®, (2) PowerSoil®+saturated phosphate buffer, (3) NucleoSpin®, and (4) NucleoSpin®+saturated phosphate buffer. Finally, each DNA extraction sample was prepared for amplicon sequencing in a one-step and two-step qPCR protocol. For both, the 1391f/EukBr assay (Stoeck et al. 2010) targetting the V9 region of the 18S rRNA gene was used. Refer to Olmedo-Rojas et al. (2021) for a complete description of the collection, processing and analysis of the samples.Sequencing results associated with this metadata are files '1_relabeled.fast.gz' and '2_relabeled.fast.gz' and can be found under the 4370 AAS project (The role of volcanoes in maintaining Antarctic terrestrial biodiversity). See Olmedo-Rojas et al. (2021) for more information on the bioinformatics analysis of both files.Sequencing reads present the following metadata headings: (1) Collection site (i.e., 'Stornes', (2) sample ID number, (3) library preparation method (i.e., 1-step' or '2-step' library), and (4) barcode combination used (e.g., AACCGA-ACGAGTC). See file 'Stornes_metadata.csv' for complete information on metadata associated with each sequencing read.

若要减缓环境变化带来的影响，解析南极陆地生物的分布模式至关重要。目前仍未得到解答的关键科学问题包括：南极的生物多样性热点区域位于何处？陆地群落究竟是生物学上相互隔离，还是存在广泛的连通性？基因组学技术的快速发展为解答上述及相关问题提供了全新契机。本研究以东南极斯托恩斯半岛（Stornes Peninsula）的土壤样品为材料，优化环境DNA（environmental DNA, eDNA）元条形码测序方案。我们比较了DNA采样、DNA提取与文库制备的多种方法，旨在开发一种低成本、低耗时的eDNA元条形码测序方案，以评估低生物量土壤样品中的动植物多样性。研究结果显示，结合省时且低成本的提取与文库制备方法，仅需少量起始材料即可获得相似度较高的操作分类单元（Operational Taxonomic Unit, OTU）数据集。此外，与其他区域相比，本次检测到的类群丰度较低。上述研究结果对未来南极陆地生物群落的监测工作具有极高的应用价值。土壤样品采集于2018年12月，采集地点为东南极斯托恩斯半岛（坐标：69°24.011'S, 76°05.382'E）。在1平方米样方的四个角分别采集约50毫升表层土壤样品。对这四份土壤样品分别进行单独处理，并设置混合处理组。随后，采用以下四种方法对每份重复样品进行DNA提取：(1) PowerSoil®试剂盒法；(2) PowerSoil®+饱和磷酸缓冲液法；(3) NucleoSpin®试剂盒法；(4) NucleoSpin®+饱和磷酸缓冲液法。最后，采用一步法与两步法实时定量聚合酶链式反应（quantitative PCR, qPCR）两种文库制备方案，对每份DNA提取样品进行扩增子测序文库构建。两种方案均采用靶向18S rRNA基因V9区的1391f/EukBr引物扩增体系（Stoeck等，2010）。有关样品采集、处理与分析的完整细节，请参阅Olmedo-Rojas等人（2021）的研究成果。本元数据对应的测序结果文件为"1_relabeled.fast.gz"与"2_relabeled.fast.gz"，可在编号为4370的澳大利亚南极署（Australian Antarctic Division, AAS）项目《火山在维持南极陆地生物多样性中的作用》中获取。有关这两份文件的生物信息学分析细节，请参阅Olmedo-Rojas等人（2021）的研究成果。测序读段对应的元数据字段包括：(1) 采样地点（即"Stornes"）；(2) 样品编号；(3) 文库制备方法（即"一步法文库"或"两步法文库"）；(4) 所用的条形码组合（例如AACCGA-ACGAGTC）。有关每条测序读段对应的完整元数据信息，请参阅"Stornes_metadata.csv"文件。