Ampure xp magnetic bead system

Due to the rapidly adaptive nature of the surface mucus layer (SML) to local environments and/or stress, bacterial genomic DNA was extracted from coral SML and seawater using the CTAB (Cetyl-trimethyl-ammonium-bromide) method [119 (link)]. To amplify the bacterial 16S rRNA gene from SML and water samples, hypervariable regions V3 and V4 of ribosomal DNA were targeted (~550pb) using 341F and 805R universal bacterial primers with an Illumina overhang adaptor (Additional file 2: Table S1) according to the manufacturer’s protocol (Illumina, San Diego, CA, USA). The PCR amplicon was cleaned by an AMPure XP magnetic bead system (Beckman Coulter, Brea, CA, USA), and 5 μl of cleaned PCR amplicon used for indexing PCR using Nextera XT V2 kit (A&B index kit) (Illumina) according to the manufacturer’s protocol. The indexed PCR amplicon was cleaned again by AMPure XP magnetic beads and then quantified using a FLUOstar Omega microplate reader (BMG Labtech, Germany) using Quant-iT PicoGreen dsDNA assay kit (Invitrogen, USA). All samples were then pooled in equimolar ratios. The quality of the final pooled library was checked on a 1% agarose gel as well as on a Bioanalyzer (Agilent 2100, Santa Clara, CA, USA). Version 3 chemistry kit was used in HiSeq and sequencing was conducted at the TGAC genomic analysis center (Norwich, UK).

PCR amplicons were cleaned up before adaptor addition using the Ampure XP magnetic bead system (Beckmann Coulter, MA, United States) according to manufacturer’s recommendation. Dual barcode indices and sequencing adaptors were attached to each amplicon using the Illumina Nextera XT Index kit (Illumina, Inc., San Diego, CA, United States) following the manufacturer’s protocol, followed by a further Ampure XP cleanup step. Purified amplicons were quantified using the Quant-iT^TM PicoGreen^TM dsDNA Assay Kit (Thermo Fisher Scientific) and a Tecan Safire microplate reader (Tecan Group, Männedorf, Switzerland). Equimolecular amounts from each individual sample in 10 mM of Tris were combined, and the pooled library was additionally purified with two rounds of Ampure XP cleanup step. The library was sequenced by the Genomics Unit at “Fundación Parque Científico de Madrid” (Madrid, Spain) using the Illumina MiSeq platform (Nano-V2; PE 2x 250 bp). The ZymoBIOMICS microbial standard (Zymo Research Corp., Irvine, CA, United States) and water (no template DNA) were used as internal positive and negative controls, respectively, for library construction and sequencing. Raw sequence data have been deposited in the Sequence Read Archive (SRA) database at the NCBI under BioProject accession number PRJNA684121.