Bacterial 16S rRNA Profiling of Acropora Corals

In 8 coral Acropora samples and 1 seawater sample collected from Kochi, regions V1–V2 of the bacterial 16S rRNA gene were amplified by PCR using the bacterial universal primers 27F and 341R (5′-CTGCTGCCTCCCGTAGG-3′). DNA tagging PCR was used to fuse unique tags to each PCR product, which was conducted as described (2 (link)). Amplicons from the 9 Kochi samples were quantified and pooled in equal amounts. Multiplex sequencing was performed with a Roche 454 GS junior with Titanium chemistry - System (Roche 454 Life Sciences, Branford, CT, USA) at Mission Biotech (Taipei, Taiwan).
Raw sequencing reads were sorted into samples according to barcodes using an in-house sorter script (http://140.109.29.21/scripts/). After sorting and trimming with data from specific primers, high-quality reads were extracted using MOTHUR (52 (link)) with the following criteria: 1) read lengths between 280 and 350 bp; 2) average quality score >20; 3) homopolymer length <8 bp; and 4) removal of reads with any ambiguous base (N). Thereafter, the 4 nucleotide tags and primer sequences were removed. Chimeric reads were inspected and eliminated by UCHIME (15 (link)) with USEARCH v7.0.1090 (parameters: reference mode, rdp_gold database, and mindiv of 3). A total of 91,211 qualified sequences were retained for subsequent analyses.