16S rRNA gene sequencing of wetland soil metagenome
Illumina compatible amplicons of the 16S V4 rRNA gene were constructed using the method described by Kozich JJ, et al., Appl Environ Microbiol. 2013 Sep;79(17):5112-20. After validation of PCR products these were divided into two pools, 48 libraries apiece. Each pool was loaded on one MiSeq flow cell (v2) and sequenced in a 2x250bp paired end format using a standard 500 cycle reagent cartridge. Custom sequencing primers complementary to the V4 target primers were used in sequencing (see above ref.). Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq.