Model 475 gradient former

Denaturing gradient gel electrophoresis (DGGE) was performed as previously described [24 (link)] using the DCode^TM Universal Mutation Detection System instrument and model 475 gradient former according to the manufacturer’s instructions (Bio-Rad Labs, Hercules, CA, USA). The V2–V3 region of the 16S rRNA genes (positions 339–539 in the Escherichia coli gene) of bacteria in gut samples was amplified with the primers HDA1-GC and HDA2. PCR reaction mixtures and the amplification program were the same as described previously [24 (link)]. The denaturing gradient was formed with two 8% acrylamide (acrylamide-bis 37.5:1) with denaturing gradients ranging from 20–80% for analysis of the amplified 16S rRNA fragments. The 100% denaturant solution contained 40% (v/v) deionized formamide and 7 M urea. PCR product (40 μL) was mixed with 40 μL dye before loading. Gels were run in 0.5× Tris/Acetate/EDTA buffer at 60 °C for 5.2 h at 180 V, 210 mA, stained with Gel Star (Lonza Japan, Tokyo, Japan) for 30 min, and analyzed by ChemiDoc MP (Bio-Rad, Hercules, CA, USA). Image Lab software, version 5.0 (Bio-Rad) was used for the identification of bands and normalization of band patterns from DGGE gels.

To determine dominant Symbiodinium types within the six species of Gulf corals, the ITS2 region was analysed by DGGE-PCR as detailed in Supplementary Information. In short, the ITS2 region was amplified from genomic DNA template by PCR using the primer pair SYM_VAR_5.8SII and SYM_VAR_Clamp. DGGE analysis was conducted using a BioRad DCode System for DGGE with a model 475 gradient former. Samples were run on a 32.5–57.5% gradient for 1400 Vh. The banding patterns produced on the gels for each sample, known as a ‘fingerprint’ are characteristic of a given Symbiodinium type or mix of types. The use of pre-characterized markers allows the identification of subcladal type according to the distance bands run in the DGGE gel (Supplementary Fig. S4). By extracting and sequencing novel bands a library of known fingerprint-type associations is built and can be used to assess future samples.

Denaturing gradient gel electrophoresis (DGGE) was carried out as previously reported [18 (link)] using a DCode^TM Universal Mutation Detection System instrument and a Model 475 gradient former according to the manufacturer’s instructions (Bio-Rad Labs, Hercules, CA, USA). The V2-V3 region of the 16S rRNA genes (positions 339 to 539 in the Escherichia coli gene) of bacteria in the gut samples was amplified by primers HDA1-GC and HDA2 as described by Walter et al. [19 (link)]. PCR reaction mixtures and the amplification program were the same as described previously [19 (link)] except that 30 amplification cycles were used. The denaturing gradient was formed using two 8% acrylamide gels (acrylamide-bis 37.5:1) with denaturing gradients ranging from 30 to 70% for analysis of the amplified 16S rRNA fragments. The 100% denaturant solution contained 40% (v/v) deionized formamide, and 7 M urea. PCR products (40 μL) were mixed with 40 μL of loading dye before loading. Gels were run in 0.5 × TAE at 60 °C for 5.2 h at 180 V, 210 mA, stained with Gel Star (Lonza, ME, USA) for 30 min, and analyzed by Chemi Doc MP (Bio-Rad Laboratories, CA, USA). Image Lab software version 5.0 (Bio-Rad) was used for the identification of bands and normalization of band patterns from DGGE gels.