48 hour biofilm of Bacillus sp. Strain C20, Accession #KY075739 after 8 hour challenge with freshwater aquatic metagenome
1-3 l of alkaline-PEG extracted DNA was used to amplify the V4 region with primers dual indexed fusion primers targeting the V4 region (515f/806r) of 16S rRNA genes. The primers without barcodes and linkers were; forward, GTGCCAGCMGCCGCGGTAA; reverse, GGACTACHVGGGTWTCTAAT (49). Amplification products were normalized (Invitrogen SequalPrep normalization plate) and cleaned with AMPure XP beads. The pool was then loaded on an Illumina MiSeq v2 flow cell and sequenced with a 500-cycle v2 reagent kit (PE250 reads) paired end format. Base calling was performed by Illumina Real Time Analysis Software (RTA) v1.18.54 and output of RTA demultiplexed and converted to FastQ files with Illumina Bcl2fastq v1.8.4.