title: Sponge Metagenome from marine sponges of India
design: Sponge metagenomic DNA was isolated using the DNeasy DNA isolation kit (Qiagen GmbH, Hilden) following the manufacturers protocol. In total, 6 samples of metagenomics DNA were subjected to Illumina Sequencing, in which 3 samples belong to Gelliodes (named as Gr1, Gr2, and Gr3) and 3 samples belong to Callyspongia (named as Red1, Red2, Red3). The purity of the DNA samples was checked spectrophotometrically using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, USA). The concentration of DNA samples was quantified using Qubit. To verify the size of PCR enriched fragments, we check the template size distribution by running on Agilent Technologies 2100 Bioanalyzer using a DNA 1000 chip. Bacterial V3-V4 hyper variable regions of the 16S rRNA genes were amplified using the specific universal primers 341F, 5-CCTACGGGAGGCAGCAG-3 and 518R, 5-ATTACCGCGGCTGCTGG-3.  PCR amplification was performed using an initial denaturing step of 98 C for 3 min, followed by 20 cycles at 98 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec, and then an elongation step of 72 C for 5 min. Subsequently, indexing PCR, Illumina sequencing adapters, and dual Indexing barcodes were added with limited PCR cycles. The prepared libraries were quantified using qPCR according to the Illumina qPCR Quantification Protocol Guide. To generate a standard curve of fluorescence readings and calculate the library sample concentration, Roche's Rapid library standard Quantification has been used. The purified PCR products (library) were pooled into equimolar ratios and paired-end reads were generated on an Illumina GAIIx sequencer. Image analysis and base calling were performed using Illumina Analysis pipeline (Version 2.2).