title: nirS metagenome
design: Thepolymerase chain reaction (PCR) was performed in 50 L reaction mixture containing 5L of 10  PCR buf-fer, 4L of MgCl2(25 mM), 3L of deoxynucleotide tri-phosphates (dNTPs, 2.5 mM each), 1L of each primer(10M), 30 ng template DNA, and 0.3LofTa qpoly-merase (5 U L-1 Fermentas). PCR cycling was carriedout in a thermocycler (Applied Biosystems Veriti Thermal Cycler) using a touchdown program: denaturationat 94 C for 5 min, 11 cycles of denaturation at 94 C for1 min, annealing at 65 C for 1 min, and extension at 72 C for 10 min.