Partial nifH sequences from cDNA isolated from environmental Trichodesmium colonies of tuft morphology collected on 14 Mar 2014 (repB)
Complimentary DNA was constructed from RNA extracts using the nifH3 gene-specific primer. Partial nifH sequences were amplified from environmental DNA using nested PCR with the universal primers of Zehr and McReynolds (1989) and Zani (2000). Samples were multiplexed using a one-step PCR with gene-specific sites, forward and reverse barcodes, Illumina linkers, and sequencing primer binding regions, similar to the method of Kozich et al. (2013). Though this was a paired-end run and both forward and reverse index reads were used for demultiplexing, only forward reads could be used for phylogenetic analyses due to the poor quality of reverse reads.