16S V4 tag sequencing of xenic Microcystis aeruginosa NIES-843
The V4 region of the 16s rRNA gene was amplified from each sample using a dual indexing sequencing strategy (74). Sequencing was done on the Illumina MiSeq platform, using a 2 x 250 MiSeq Reagent Kit V2 (Illumina cat# MS-102-2003), according to the manufacturer's instructions with modifications found in the Schloss SOP (https://github.com/SchlossLab/MiSeq_WetLab_SOP). Accuprime High Fidelity Taq (Life Technologies cat # 12346094 ) was used instead of the Accuprime Pfx supermix. PCR products were visualized using an E-Gel 96 with SYBR Safe DNA Gel Stain, 2% (Life technologies cat# G7208-02).  Libraries were normalized using a SequalPrep Normalization Plate Kit (Life technologies cat # A10510-01) following the manufacturer's protocol for sequential elution.  The concentration of the pooled samples was determined using a Kapa Biosystems Library Quantification kit for Illumina platforms (KapaBiosystems KK4824).  The sizes of the amplicons in the library were determined using the Agilent Bioanalyzer High Sensitivity DNA analysis kit (cat# 5067-4626).  The final library consisted of equal molar amounts from each of the plates, normalized to the pooled plate at the lowest concentration. Library Preparation for Sequencing and Sequencing Libraries were prepared according to Illuminas protocol for Preparing Libraries for Sequencing on the MiSeq (part# 15039740 Rev. D) for 2nM or 4nM libraries. If the library concentration was below 1nM an alternative method was used for denaturation (75). PhiX and genomes were added in 16S rRNA gene amplicon sequencing to create diversity. Sequencing reagents were prepared according to the Schloss SOP, custom read 1, read 2 and index primers were added to the reagent cartridge. FASTQ files were generated for paired end reads.