2.3 DNA extraction and 16s rRNA amplicon sequencing
DNA from sediment and sea cucumber (skin and gut) samples was extracted using the DNeasy PowerLyzer PowerSoil Kit (Qiagen, Germantown, MD). DNA from seawater samples was extracted using the DNeasy PowerWater Kit (Qiagen, Germantown, MD), following the manufacturer’s instructions. In both cases, the same batch of DNA extraction kits was used for all the samples. Overall, we followed guidelines for sequence-based analyses of microbial communities (Eisenhofer et al., 2019) to avoid any potential contamination of samples originating from reagents or the laboratory environment. As part of this, DNA extractions were performed in the Marine Molecular Lab (HKU) under a sterilised laminar flow hood. After extractions, DNA concentrations were verified using BioDrop (Biochrom, UK), and DNA quality was checked via agarose gel electrophoresis. Total genomic data was submitted to Novogene Bioinformatics Technology Co., Ltd., Beijing, China for amplicon sequencing. The V3-V4 hypervariable region of the 16S ribosomal RNA gene was amplified with the primers 341F (5’- CCTACGGGNGGCWGCAG-3’) and 806R (5’- GGACTACNNGGGTATCTAAT -3’) (Y. Yu et al., 2005). PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs, US) following the manufacturer’s instructions. Amplicons from different samples were mixed in equidensity ratios and purified with the Qiagen Gel Extraction Kit (Qiagen, Germany). NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, US) was used to construct DNA libraries (paired-end), following the manufacturer’s recommendations. Index codes were added, and the library quality was assessed on the Qubit® 2.0 Fluorometer (Thermo Scientific, US) and 2100 Bioanalyzer system (Agilent, US). Amplicons from different samples were mixed in equimolar amounts and sequenced on the Illumina NovaSeq 6000 platform with sequencing strategy PE250. Two negative controls were included during library preparation to check potential contamination. However, these controls were not sequenced as they did not generate any product during PCR amplification. Primary processing was done by removing barcodes, adaptor sequences and indices.