High-throughput sequencing of comammox Nitrospira amoA genes
For amplicon sequencing, the amoA gene of comammox Nitrospira was amplified using primers Ntsp-amoA 162F/Ntsp-amoA 359R, and the forward primers were modified to contain a unique barcode (10 bp) at the 5 end for each sample. PCR reactions were performed in a 50 l mixture containing 25 l of 2  Rapid Taq Master Mix (Vazyme, Nanjing, China), 2 l of each primer (10 M) and 20 ng of template DNA. The amplification conditions were as follows: 95C for 3 min, and 30 cycles of 95C for 15 s, 48C for 30 s, and 72C for 30 s, followed by 72C for 5 min. Each sample was amplified in triplicate, and the PCR products were purified using AxyPrep gel extraction kit (AxyGen, Hangzhou, China) and quantified by Nano-Drop ND-1000 spectrophotometer. Purified amplicons were pooled together in equimolar ratios into one single tube and sent for paired-end sequencing (2  150 bp) on an Illumina HiSeq platform (Illumina, San Diego, United States) at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.