In the past, studies on the effects of different stresses on algal epiphytic bacteria were mostly conducted in aseptic systems and isolation cultures and tried to identify epiphytic bacteria related to macroalgae. However, because sterile experimental systems are difficult to obtain and culturable bacteria constitute less than 1% of the bacteria present in nature, in actual algal environments, bacteria do not exist in isolation61 (link); thus, these methods are cumbersome, and the information obtained is not accurate or comprehensive. In this paper, high-throughput sequencing, which is relatively fast and has a relatively low cost and workload23 (link), was used to extract DNA samples directly from algae.
On the ultra-clean platform, the bacterial suspension obtained in was filtered through sterile gauze to remove any impurities, and then the bacteria were filtered and collected on a 0.22 µm filter membrane using a vacuum filtration device. DNA was extracted from these membranes using an E.Z.N.A. Stool DNA Kit (Omega Bio-tek, USA) following the manufacturer’s instructions. At the same time, a negative control was used to determine the contamination from the DNA Kit. The 16S rDNA V3-V4 region was amplified via PCR using 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTATCTAAT-3′) primers. The purified amplicons were subsequently sequenced (PE250) on the Illumina Hiseq 2500 platform according to standard protocols by Guangzhou Genedenovo Biotechnology Co., Ltd.
On the ultra-clean platform, the bacterial suspension obtained in was filtered through sterile gauze to remove any impurities, and then the bacteria were filtered and collected on a 0.22 µm filter membrane using a vacuum filtration device. DNA was extracted from these membranes using an E.Z.N.A. Stool DNA Kit (Omega Bio-tek, USA) following the manufacturer’s instructions. At the same time, a negative control was used to determine the contamination from the DNA Kit. The 16S rDNA V3-V4 region was amplified via PCR using 341F (5′-CCTACGGGNGGCWGCAG-3′) and 806R (5′-GGACTACHVGGGTATCTAAT-3′) primers. The purified amplicons were subsequently sequenced (PE250) on the Illumina Hiseq 2500 platform according to standard protocols by Guangzhou Genedenovo Biotechnology Co., Ltd.
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