title: nifH amplicon sequencing of surface (5-200 m) water samples across a West to East transect(~14 °N) of the Tropical North Atlantic.
design: Extracted DNA samples were prepared for nifH amplicon sequencing using a nested PCR following established protocols (Zehr et al., 2011) with modifications as previously described ( Bentzon-Tilia et al., 2015). Illumina indices were added to amplicons in the second PCR round. In addition to the nifH1 and nifH2 primer sequences, primers contained a linker sequence, an 8-base barcode, and the Illumina specific region P5 (forward primer) or P7 (reverse primer). Refs: Zehr, J. P. & Turner, P. J. in Methods in Microbiology Vol. Volume 30    271-286 (Academic Press, 2001); Bentzon-Tilia, M. et al. Significant N2 fixation by heterotrophs, photoheterotrophs and heterocystous cyanobacteria in two temperate estuaries. The ISME journal 9, 273-285 (2015)