title: 16S amplicons reads of microbiomes from coral reef samples collected in the Southern Caribbean Sea, North Colombian shore
design: 16S amplicon sequencing from microbiomes in coral reef components in two close locations at the Southern Caribbean Sea. Small amounts >10 grams of the specimens were collected by SCUBA diving in the coordinates referenced, placed in tubes with 2.5 volumes of absolute ethanol and kept at 4C until further processing. Total metagenomic DNA extraction was performed using a DNA isolation kit (Product # 27600, Norgen Biotek Corporation, Thorold, ON, Canada) according to the manufacturers instructions. Total purified DNA was eluted using the Elution Buffer B in a sterile microcentrifuge tube and was stored at -20C until further amplification and sequencing. DNA was quantified using Nanodrop 2000 (Thermo Fisher Scientific, USA). The purified metagenomic DNA extracts from the samples were used as templates on independent PCR reactions targeting two hypervariable regions of the bacterial/archaeal 16S rRNA genes with primers annealing in highly conserved delimiting regions (sequences in 5'-3') forward primer 341F CCTAYGGGRBGCASCAG and reverse primer 806R GGACTACNNGGGTATCTAATT including in 5' a unique barcode for multiplexing. The total volume of the PCR was 20 ul, using 10 ng as DNA template with 10 l of Phusion High-Fidelity PCR Master Mix (New England Biolabs) and 0.2 pmol/l of forward and reverse primers. Thermocycling conditions were as follows: initial denaturation at 98C for 1 min, 30 cycles of 98C for 30 s, annealing at 55C for 30 s, elongation at 72C for 1 min, and a final extension of 72C for 5 min. The amplified products were then checked and quantified using agarose DNA electrophoresis and Qubit 2.0 Fluorometer (Thermo Scientific). Sequencing libraries were generated using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, USA) and index codes were added. The library quality was checked using an Agilent Bioanalyzer 2100 system. The samples were sequenced using a Novaseq using 2 x 250 cycles and the demultiplexed split datasets of the paired-end reads for each sample were generated on the fly.