16S rRNA sequence of NoIC 2
16S rDNA gene amplicons were obtained following the 16S rDNA gene Metagenomic Sequencing Library Preparation Illumina protocol (Cod. 15044223 Rev. A). The genespecific sequences used in this protocol target the 16S rDNA gene V3 and V4 region. Illumina adapter overhang nucleotide sequences are added to the genespecific sequences. The primers are selected from the Klindworth et al. (2013). The full length primer sequences, using standard IUPAC nucleotide nomenclature, to follow the protocol targeting this region are: 16S rDNA gene Amplicon PCR Forward Primer = 5'. TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG. 16S rDNA gene Amplicon PCR Reverse Primer = 5'. GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC. We used microbial genomic DNA (5 ng/l in 10 mM Tris pH 8.5) to initiate the protocol. After 16S rDNA gene amplification, the mutiplexing step was performed using using Nextera XT Index Kit (FC-131-1096). We run 1 l of the PCR product on a Bioanalyzer DNA 1000 chip to verify the size, the expected size on a Bioanalyzer trace is ~550 bp. After size verification the libraries were sequenced using a 2x300pb paired-end run (MiSeq Reagent kit v3 (MS-102-3001)) on a MiSeq Sequencer according to manufacturers instructions (Illumina).