DOI: 10.6084/m9.figshare.20308905
title: Fastq data

description: 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.