Fastq data

1 Material and Methods 1.1 eDNA seawater sample collection The eDNA seawater samples were collected from 40 sampling sites across Indonesia (Fig. 1). Seawater samples were collected from the surface at a depth of about 1 meter. A 3L volume of each seawater sample was then filtered through a 0.45 µM Pall Corporation sterilized filter paper using a vacuum pump to draw the water through the filter. After the filtering process was complete, the filter paper was then cut into two halves using sterilised scissors. Each half was placed into a 2 mL cryotube filled with 1 mL DNA shield (ZymoBIOMICS DNA/RNA shield). Contamination was prevented through the strict sterilisation of all the sampling equipment used at each stage of the sampling procedure with a 30% solution of commercial bleach. On each sampling site, a negative control using sterilized ddh2o was used to filtering at the end of sampling section to monitor any contamination according to West et al. (2020, 2021). 1.2 eDNA laboratory analysis, library preparation and next generation sequencing The eDNA retained in the filter papers was extracted using Geneaid gSYNC™ DNA Extraction Kits following the manufacturer’s protocol. The first PCR amplified the target region using 16S rRNA MiDeca Primers (Forward and Reverse) (Komai et al., 2019) with connecting adapters: 5'-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG (forward sequence adapter) and 5'-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G (reverse sequence adapter). The primers target a hypervariable region of the 16S rRNA gene (154 – 189 bp) which contains sufficient information to identify Decapods to taxonomic family, genus and species (Komai et al., 2015). To date, primer selection to identify organism based on eDNA metabarcoding by using the hypervariable regions are suitable targets given their sequence variation enables for strong taxonomic resolution in macroeukaryotes (Miya et al. 2015; Berry et al. 2017; Stat et al., 2017; Jeunen et al., 2019). The MiDeca itself is universal primer to amplify from 56 families, 126 genera, and 207 species (Komai et al., 2019). The first PCR reaction volume was 25 μl, consisting of 13 µL KAPA Hifi Hotstart Readymix, 1 µL each of 1 nM primers (Forward and Reverse), 4 µL ddH2O, and 7 µL DNA Template. The DNA amplification PCR profile stages included: (1) pre-denaturation of the template DNA at 95°C for 5 minutes; (2) denaturation of the template DNA at 98°C for 10 seconds; (3) annealing at 60º C for 10 seconds; (4) primary extension at 72°C for 10 seconds and (5) final extension (post extension) at 72°C for 5 minutes with 35 cycles of stages (2)-(4). Two negative controls (i.e. blank template) were used when running the 96 Universal peqStAR PCR machine (Peqlab Ltd, USA) in order to check for contamination. The PCR product quality was visualised through electrophoresis on 2% agarose gel (100 mL 1X TAE buffer and 2 g agarose) run at 100 Volts for 38 minutes. The results were visualized using UV fluorescence via an Alphaimager Mini Gel Documentation System (ProteinSimple Ltd, California, USA) All PCR products which passed the electrophoresis quality control underwent a second PCR for indexing purposes. The IDT double index and Illumina sequencing adapter for Illumina - Nextera DNA Unique Dual Index, Set A (catalogue number 20027213) (Illumina, San Diego, USA) were added to the target amplicon in the second PCR, using 12.5 μl of Kapa HotStart HiFi 2 × ReadyMix DNA polymerase (Kapa Biosystems Ltd., London, UK) and 2 μl of PCR product. The PCR cycle comprised an initial denaturation at 95°C (3 minutes), then 9 cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 5 minutes. The first PCR and second PCR products were purified using AMPure XP beads (Beckman Coulter, Inc) before proceeding to the next step. DNA sequencing was performed on an Illumina iSeq 100 using the standard reagent kit and cycles following the modified protocol of Illumina MiSeq 16S metagenomic sequencing library protocol. The concentration of each amplicon barcode library was assayed using a Qubit fluorometer and diluted to 10 nM before the libraries were pooled. The pooled library was diluted and denatured according to the Illumina MiSeq library preparation guide. Aliquots of 16 µL of the 40 pM amplicon library and 4 µL of the 60 pM PhiX Illumina version 3 control library were pooled as the final product. The Illumina iSeq v.2 Reagent kit for 2×150 bp PE was used with a run-time of about 18 hours and produced a Fastq file. The specific barcode index of the IDT double index and the Illumina sequencing adapter for Illumina - Nextera DNA Unique Dual Index were excluded during the process.

1 材料与方法
1.1 环境DNA（eDNA）海水样本采集
本研究从印度尼西亚境内的40个采样点采集环境DNA（eDNA）海水样本（图1）。海水样本采集自表层约1米水深处。随后使用真空泵抽滤，将每份3L体积的海水样品通过0.45 μm的颇尔（Pall Corporation）除菌滤膜进行过滤。过滤完成后，使用无菌剪刀将滤膜剪成两半。将每一半滤膜分别放入装有1 mL DNA保存液（ZymoBIOMICS DNA/RNA shield）的2 mL冻存管中。为避免污染，在采样各阶段均使用30%商用漂白剂溶液对所有采样设备进行严格灭菌处理。按照West等人（2020、2021）的方法，每个采样点在采样结束时均设置使用无菌双蒸水（ddH₂O）的过滤阴性对照，以监测污染情况。

1.2 环境DNA实验室分析、文库制备与高通量测序
使用Geneaid gSYNC™ DNA提取试剂盒并按照制造商说明书提取滤膜上留存的eDNA。首轮PCR采用带有连接接头的16S rRNA MiDeca引物（正向、反向）（Komai等，2019）扩增目标区域，正向序列接头为5'-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG，反向序列接头为5'-GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G。该引物靶向16S rRNA基因的高变区（154~189 bp），该区域具备足够的序列信息，可将十足目（Decapods）物种鉴定至科、属、种水平（Komai等，2015）。迄今为止，基于环境DNA元条形码（metabarcoding）的物种鉴定中，选用高变区作为靶标引物是较为合适的选择，因其序列变异度可在大型真核生物中实现良好的分类学分辨率（Miya等，2015；Berry等，2017；Stat等，2017；Jeunen等，2019）。MiDeca引物为通用引物，可扩增56科、126属、207个物种的靶标序列（Komai等，2019）。
首轮PCR反应体系总体积为25 μL，包含13 μL KAPA HiFi HotStart 预混液、1 μL（1 nM）正向及反向引物各1份、4 μL 无菌双蒸水（ddH₂O）以及7 μL DNA模板。DNA扩增的PCR反应程序如下：（1）模板DNA预变性：95℃下维持5分钟；（2）变性：98℃下10秒；（3）退火：60℃下10秒；（4）链延伸：72℃下10秒；（5）最终延伸：72℃下5分钟。其中步骤（2）~（4）循环35次。使用96孔Universal peqStAR PCR仪（Peqlab有限公司，美国）进行扩增时，设置2个阴性对照（即空白模板对照）以检测污染情况。
通过琼脂糖凝胶电泳对PCR产物的质量进行检测：配制2%琼脂糖凝胶（100 mL 1×TAE缓冲液+2 g琼脂糖），以100 V电压电泳38分钟，随后使用Alphaimager Mini凝胶成像系统（ProteinSimple有限公司，美国加利福尼亚州）通过紫外荧光成像读取结果。所有通过电泳质量质控的PCR产物均需进行第二轮PCR以添加索引序列。
第二轮PCR中，使用IDT双索引与Illumina Nextera DNA Unique Dual Index试剂盒A套装（目录号20027213，Illumina公司，美国圣地亚哥）的测序接头对靶标扩增子进行索引添加，反应体系包含12.5 μL Kapa HotStart HiFi 2×预混DNA聚合酶（Kapa Biosystems有限公司，英国伦敦）以及2 μL首轮PCR产物。第二轮PCR反应程序为：95℃初始变性3分钟，随后进行9个循环：95℃变性30秒、55℃退火30秒、72℃延伸30秒，最终于72℃下延伸5分钟。首轮及第二轮PCR产物均使用AMPure XP磁珠（贝克曼库尔特有限公司）进行纯化，随后进行后续实验步骤。
参照Illumina MiSeq 16S宏基因组（metagenomic）测序文库制备方案并进行优化，使用Illumina iSeq 100测序平台及配套标准试剂试剂盒与测序循环完成DNA测序。使用Qubit荧光计对每个扩增子条形码文库的浓度进行定量，将其稀释至10 nM后进行文库混合。混合后的文库按照Illumina MiSeq文库制备指南进行稀释与变性处理。取16 μL 40 pM的扩增子文库与4 μL 60 pM的PhiX Illumina V3对照文库混合，作为最终上机文库。使用Illumina iSeq v.2 2×150 bp双端测序试剂试剂盒，测序运行时长约18小时，最终产出Fastq格式文件。本研究在实验流程中未使用IDT双索引与Illumina Nextera DNA Unique Dual Index试剂盒自带的特异性条形码索引接头。