title: Profiling of bacterial communities present in coral Acropora (palk bay) by 16S rRNA gene based metagenomic analysis.
design: the coral samples preserved in DESS solution were washed with sterile deionized water to remove excess salt debris and in aseptic condition, the coral tissues were removed by controlled flushing of sterile air over the coral surface with the use of an air compressor and a delivery gun. The coral tissues removed from the coral skeleton were dispensed as aliquots and then stored at -20C until further processing. Prior to isolation of total DNA, the aliquoted samples were thawed at 4C and subjected to total DNA isolation using Plant DNeasy DNA isolation kit (Qiagen GmbH, Hilden) following the manufacturers protocol. The DNA samples obtained from independent extractions from different samples were analyzed by agarose gel electrophoresis. The purity of the DNA samples was checked spectrophotometrically using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, USA). Amplification of 16S rRNA gene was performed by using universal bacterial 16S rRNA (V3-V4 region) gene primers:  338F (5'- ACTCCTACGGGAGGCAGCA-3') and 806R (5'- GGACTACHVGGGTWTCTAAT-3'). PCR reaction mixture (50 L) is composed of 50-100 ng of DNA sample, 0.2 mM of each of the primers (338F and 806R), 5X TransStart Fast pfu Buffer, 2.5mM dNTPs, TransStart Fastpfu DNA polymerase and double distilled water to make up the final volume to 50 L. Amplification conditions of the pre-experimental PCR were initial denaturation at 95 for 2 mins, then 30 cycles of denaturation at 95 for 20 s, annealing at 55 for 30 seconds, extension at 72 for 20 seconds and final extension at 72 for 5 mins. The amplicons were electrophoresed in 2% agarose gel to detect the desired product size of 380-400 bp. Then the amplicons were subjected to bead purification before proceeding to adaptor ligation process which includes a PCR reaction with reaction mixture composed of 50 ng DNA mix, 2 L UAP1 and 12.5 L of GoldStart Master Mix and final volume made up to 50 L by adding double distilled water. PCR reaction conditions include initial step of denaturation at 94 for 5 mins, followed by 5 cycles of denaturation at 94 for 20 s, annealing at 65 for 30 seconds, extension at 72 for 1 min and final extension at 72 for 10 mins. These amplicons were again electrophoresed and subjected to bead purification to ensure ligation of adaptor sequences. Then the samples were subjected to quantification by Qubit 3.0 detection and stored at 4C or -20C.NGS sequencing and Quality filtering Amplicons were subjected to paired-end sequencing in Illumina MiSeq platform to generate 300 bp paired-end raw reads, and then pretreated. Specific processing steps are as follows: Paired-end reads were assigned to a sample by their unique barcode, and the barcode and primer sequence were then truncated. Paired-end reads were merged using FLASH, a very fast and accurate analysis tool to merge pairs of reads when the original DNA fragments are shorter than twice of the reads length. The obtained splicing sequences were called raw tags. Quality filtering was then performed on the raw tags under specific filtering conditions of Trimmomatic v0.33 quality control process (quality score more than 30). After filtering, high-quality clean tags were obtained. The tags were compared with the reference database using UCHIME algorithm to detect chimeric sequences, and then the chimeric sequences were removed. The effective tags were obtained finally.