Diazotroph community succession during the VAHINE mesocosm experiment (New Caledonia Lagoon)
Partial nifH fragments (ca. 360 base pairs) were amplified using a nested PCR assay and universal nifH primers nifH1-4, as described in Turk-Kubo et al., (2014). Two lagoon samples (day 1 and day 22), and three samples from each mesocosm (day 3, day 23, and the day where UCYN-C abundances were beginning to increase, i.e. days 11-15, see discussion below) were chosen for analysis. For each sample, triplicate PCR reactions were pooled. Internal primers were modified 5’ common sequence (CS) linkers (CS1_nifH1F: 5’-ACACTGACGACATGGTTCTACATGYGAYCCNAARGCNGA, CS2_nifH2R: 5’-TACGGTAGCAGAGACTTGGTCTADNGCCATCATYTCNCC) to facilitate library preparation at the DNA Services (DNAS) Facility at the University of Illinois, Chicago, using the targeted amplicons sequencing (TAS) approach described in Green et al. (2015). These libraries were pooled with other libraries to achieve a target depth of ca. 40,000 sequences per sample. Sequencing of paired end reads was performed at the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign using Illumina MiSeq technology.