title: Microbial Diversity in oil reservior
design: Extraction of genome DNA For biofilm microbial community analysis, in each system was scrapped and analysed using Mi sequencing. The biofilm total genomic DNA was extracted using a bacterial genomic DNA kit according to the manufacturer's instructions. The extraction was carried out triplicate. To check the purity and quantity of nucleic acids carried out- Nuclease free water: 1  l   and aqueous solutions of nucleic acids: 1  l   by NanoDropTM 2000 Spectrophotometer and additionally we monitored with 1% agarose gels. Qubit . 2.0 Fluorometer- It is used to check the exact concentration of dsDNA present in the extracted DNA    Amplicon generation V3 region of the 16S rRNA gene amplicon primers 314F (5'CCTACGGGAGGCAGCAG3' ) and 518R (5'ATTACCGCGGCTGCTGG 3 ), the genes PCR to amplify template out of a DNA sample using region of interest specific primers with overhang adapters attached. All PCR reactions were carried out in 25  l reactions following reaction buffer (5x concentration)   5 l, 10mM each dNTP 0.4 l, DNA polymerase (2000U/ml) 0.2 l, V3 (10pM) 2 l of forward and reverse primers, and about 10 ng template DNA. PCR in a thermal cycler using the following program:  PCR Reaction: 98 C for 30 sec ,30 cycles of: 98 C for 10 sec, 72 C for 30 sec, Extension at 72 C for 5 sec followed by 4 C hold     Amplicon selection and PCR Clean Up Run the PCR product on 2% agarose gel with SYBR  Safe DNA gel stain (10 l/100ml) in it. Size selection using gel extraction The excised gel should be between 354 to 362 bp. Purify desired amplicon DNA by using invitrogenTM  PureLinkTM Quick Gel Extraction and PCR Purification Combo Kit.   Library preparation and sequencing The library was then run on Illumina MiSeq utilizing a 300 cycle V2 Illumina Mi Seq Kit and custom primers for Sequencing according manufacturer's recommendations.  Library was sequenced on an Illumina MiSeq platform and 250 bp/300 bp paired-end reads were generated.   Data analysis This section data analysis was similarly adopted from previous publication