V4-16S-rRNA-Porites-cylindrica-white-syndrome-sample-LP131
Healthy and diseased coral microbiome composition was determined by the variable region 4 (V4) region of 16S rRNA gene amplicon Next Generation Illumina sequencing. First, DNA extractions were carried out using the Zymobiomics DNA extraction kit (ZymoResearch). PCRs for the V4 region of the 16S rRNA gene were amplified using modified forward 515F (Parada et al 2016) and reverse 806R (Apprill et al 2015) primers. The product from the first PCR was used in the second PCR designed to complete the addition of Illumina adapters and the 6-mer dual-indices as previously described by (Kozich et al 2013). Both PCRs were generated using Takara PCR buffer and Hot Start polymerase (Takara). The first PCR was carried out in 30 l volume reactions with the components at the following final concentrations: dNTPs at 0.8 mM, each primer at 0.5 M, and 30 to 100 ng of total DNA. The first PCR cycling conditions were as follows: hot start activation step for 2 min at 95 C, followed by 30 cycles of 95 C for 40 s, 58 C for 2 min, and 72 C for 1 min with a final extension of 5 min at 72 C. The second PCR was carried out in a 20-l volume reaction using the same components and final concentration as above and 30 ng of the PCR-1 product as a template. The second PCR cycling condition was as follows: a hot start activation at 95 C for 2 min, followed by five cycles of 95 C for 40 s, 59 C for 2 min, and 72 C for 1 min, and finished by a final extension of 72 C for 7 min. All samples were pooled, and gel purified using the Qiagen gel purification kit.