Metagenome V3 amplicon sequencing of the microbiome of cyanobacteria Synechocystis-SIS isolated and enriched from River mouth surface water sample,Kerala, India.
DNA was extracted from actively growing cyanobacteria culture; SIS grown in ASN media using NucleoSpin Soil DNA Extraction Kit (MACHEREY-NAGEL GmbH & Co. KG, Germany).  The extracted DNA was quantified using NanoDrop (Thermo Scientific, USA) spectrophotometer. The amplicon sequencing libraries were all prepared and sequenced at Scigenom Pvt Ltd Kerala, India. Briefly, A region of the 16S rRNA gene covering the hyper variable V3 was amplified by PCR using bacterial-specific 341F-(5' CCTACGGGAGGCAGCAG 3) and 518R-(5' ATTACCGCGGCTGCTGG 3’).  PCR reactions were performed with Phusion HF DNA polymerase (0.4U) and dNTPs (10mM each) from New England Biolabs and 10 μM of each primer and 10 ng of sample DNA. The reaction mixture was held at 98 °C for 30 sec  followed by 30 cycles of 98 °C for 10 s, , 72 °C for 30 s, extension at at 72 °C for 5 sec. Amplicons of the correct size were purified using  PureLink Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen) following manufacturers’ instructions. A second PCR was performed with primers that has Illumina indexed bar code sequences. PCR Master Mix contain 2 μL each 10 pmol/ul forward and reverse primers, 1 μL of 40mM dNTP, 10 μL of 5 X Phusion HF reaction buffers, 0.4 μL of 2U/ul F-540Special Phusion HS DNA Polymerase, 10 μL (minimum5ng) of PCR1 amplicon and water to make up the total volume to 50 μL. PCR was performed in the following conditions: 98°C for 30 sec ,15 cycles of: 98°C for 10 sec , 72°C for 30 sec , Extension at 72°C for 5 sec .The size selection was performed with Pure link Gel extraction kit (Invitrogen). Purified products were quantified using a Qubit (Invitrogen) The library preparation was done using Illumina TruSeq DNA library preparation LT Kit. Library validation is done on 2200 TapeStation Instrument. The library was then run on Illumina MiSeq utilizing a 300 cycle V2 Illumina MiSeq Kit and custom primers for Sequencing.