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.