For metabarcoding purpose, the first step involves the amplification by PCR of a given gene region (for example V4 or V9 region of 18S rRNA gene) or gene itself if its size does not exceed 600bp (the longest fragment size that can be sequenced by Illumina technology). The defined forward and reverse primers that are complementary upstream and downstream of the region of interest, needs to be designed with overhang adapters which will be used in a subsequent limited‐cycle amplification step, in order to add the dual‐index barcodes and Illumina flow cell adapters. To design illumina primers, it will be necessary to know the sequencing method, and therefore the adapters sequence. The following protocol explains the generation of nifH PCR amplicons.