V3V4 16S amplicon sequencing of reef bacterioplankton: long term mesocosms
Amplicon libraries targeting the 16S SSU rRNA V3-V4 hypervariable regions were generated from a single round of PCR using dual index 341F and 785R primers (A. Klindworth, et al., Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41, e1e1 (2013)) and 1 L genomic DNA template following standard reagents and PCR and library pooling conditions outlined by (J. J. Kozich, S. L. Westcott, N. T. Baxter, S. K. Highlander, P. D. Schloss, Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform. Appl. Environ. Microbiol. 79, 51125120 (2013)). A Zymobiomics mock community (Zymo D6306) was included as a positive control while PCR grade water was used as a negative control. Libraries were sequenced using an Illumina MiSeq V3 600 cycle run at the University of Hawaii at Mnoa Advanced Studies in Genomics, Proteomics and Bioinformatics core facility. Raw sequencing reads were processed using mothur v1.41.3.