Mullus surmuletus environmental DNA intraspecific metabarcoding Next-Generation Sequencing data

Four 250-liter aquariums were bleached clean one day prior to be used (filled with seawater; fish transfer) in Montpellier (France). Seawater collected by the French Research Institute for Exploitation of the Sea at Palavas-les-Flots (France) was first stored in a 1,000 L tank for two weeks, under UV treatment to avoid any contamination. The aquariums were then filled with 120 L of this water. Each aquarium had a closed-circuit water circulation and was equipped with an air bubbles exhauster in a tube that brought up the water on a neutral synthetic foam filter. The aquariums were thus oxygenated and the coarsest suspended matter was filtered out. The remaining seawater in the tank was used as a negative control (Aquarium 1). Nine to eleven fish were added to each of the four aquariums (Fig. 1). The aquarium water was sampled six hours after introducing the fish into the aquariums using an Athena peristaltic pump (SPYGEN, Le Bourget-du-Lac, France) with a nominal flow of 1.0 L/min to filter 30 L, and VigiDNA 0.22 μm crossflow filtration capsules (SPYGEN) with disposable sterile tubing. After filtration, 80 mL of CL1 conservation buffer (SPYGEN) was added before storing the samples at ambient temperature. We reanalyzed here two eDNA samples of 30 L replicate each, collected in the Mediterranean Sea, at Banyuls (France, coordinates: 42.41568, 3.17110) and Calvi (France, coordinates: 42.62964, 8.89161) published in a previous metabarcoding analysis and known to contain <em>M. surmuletus</em> sequences (detected with the metabarcode teleo 12S) (Boulanger <em>et al.</em> 2021). These two Mediterranean eDNA samples were amplified and sequenced using the primers developed for this study and then analyzed using the best-performing pipeline as determined by our evaluation. These two samples were used as proof of concept of the possibility to estimate within site variability in real conditions. DNA extraction and amplification from eDNA samples were performed by the company SPYGEN (Le Bourget du Lac, France) in separate, dedicated rooms following the protocol described by Polanco Fernández <em>et al.</em> (2020). The amplification was performed in a final volume of 25 μL including 1 U of AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA, USA), 10 mM of Tris-HCl, 50 mM of KCl, 2.5 mM of MgCl2, 0.2 mM of each dNTP, 0.2 μM of each primer, 0.2 μg/μL of bovine serum albumin (Roche Diagnostics, Basel, Switzerland) and 3 μL of DNA template. The PCR mixture was denatured at 95°C for 10 min, followed by 50 cycles of 30 s at 95°C, 30 s at 47°C and 1 min at 72°C and a final elongation step at 72°C for 7 min. The primers were 5’-labelled with an eight-nucleotide tag unique to each DNA sample, allowing each sequence to be assigned to the corresponding sample during the sequence analysis. Twelve replicate PCRs were run per sample. Two libraries were prepared using the MetaFast protocol (Fasteris 2020, https://www.fasteris.com/dna/) and the sequencing was performed by Fasteris (Geneva, Switzerland) on two separate runs on an Illumina MiSeq (2x250 bp) (Illumina, San Diego, CA, USA) and the Miseq Kit v3 (Illumina) following the manufacturer’s instructions. Two negative extraction controls and one negative PCR control (12 replicates of ultrapure water) were amplified and sequenced to monitor for possible contaminants (Polanco Fernández <em>et al.</em>, 2020).法国蒙彼利埃的4个250升水族箱于实验前1天完成漂白清洁，用于注入海水开展鱼类转移实验。由法国海洋开发研究院（French Research Institute for Exploitation of the Sea）于法国帕拉瓦莱弗洛采集的海水，先被储存在1000升水箱中两周，并通过紫外线处理以避免污染。随后向每个水族箱注入120升该海水。每个水族箱均配备闭路水循环系统，且在提水管路中安装了气泡排出装置，水流会流经中性合成泡沫过滤器，此举可为水体增氧并滤除最粗大的悬浮颗粒物。水箱中剩余的海水被用作阴性对照（即1号水族箱）。向4个水族箱分别投放9~11尾鱼类（见图1）。投放鱼类6小时后，使用额定流量为1.0升/分钟的Athena蠕动泵（peristaltic pump，SPYGEN公司，法国勒布尔热迪拉克）过滤30升水族箱水体，配套使用带有一次性无菌管路的VigiDNA 0.22μm错流过滤胶囊（crossflow filtration capsules，SPYGEN公司）进行采样。过滤完成后，加入80毫升CL1保存液（CL1 conservation buffer，SPYGEN公司），随后将样品置于室温下保存。 本研究重新分析了两份30升平行环境DNA（eDNA）样品，这两份样品采集自地中海法国巴纽尔斯（坐标：42.41568, 3.17110）与卡尔维（坐标：42.62964, 8.89161）海域，已在既往元条形码（metabarcoding）分析中发表，且被检出含有<em>M. surmuletus</em>序列（通过元条形码teleo 12S检测得到）（Boulanger等，2021）。使用本研究开发的引物对这两份地中海环境DNA样品进行扩增与测序，随后采用本研究评估得出的最优分析流程进行数据解析。这两份样品被用于验证在真实环境中估算位点内变异性的概念可行性。 环境DNA样品的DNA提取与扩增工作由SPYGEN公司（法国勒布尔热迪拉克）在独立专用实验室中完成，实验流程参照Polanco Fernández等（2020）发表的方案。扩增反应总体积为25微升，反应体系包含1单位AmpliTaq Gold DNA聚合酶（AmpliTaq Gold DNA Polymerase，应用生物系统公司，美国加利福尼亚州福斯特城）、10mM三羟甲基氨基甲烷盐酸盐（Tris-HCl）、50mM氯化钾（KCl）、2.5mM氯化镁（MgCl2）、每种脱氧核糖核苷三磷酸（dNTP）0.2mM、每条引物0.2μM、0.2μg/μL牛血清白蛋白（bovine serum albumin，罗氏诊断公司，瑞士巴塞尔）以及3微升DNA模板。聚合酶链式反应（PCR）反应程序为：95℃预变性10分钟；随后进行50个循环，每个循环包含95℃变性30秒、47℃退火30秒、72℃延伸1分钟；最后于72℃终延伸7分钟。所有引物的5’端均标记有一段针对每个DNA样品的独特8核苷酸标签，以便在序列分析阶段将测序读段分配至对应样品。每个样品设置12个平行PCR反应。 采用MetaFast实验方案（Fasteris，2020，https://www.fasteris.com/dna/）构建两个测序文库，随后由Fasteris公司（瑞士日内瓦）在Illumina MiSeq测序平台（2×250bp读长，Illumina公司，美国加利福尼亚州圣迭戈）上进行两次独立测序，测序试剂采用MiSeq V3试剂盒（Illumina），操作严格遵循厂商说明书。设置两份提取阴性对照与一份PCR阴性对照（12个超纯水平行反应），并对其进行扩增与测序，用于监测潜在的污染风险（Polanco Fernández等，2020）。