title: Nitrifying biofilm
design: DNA extract was fragmented to an average size of about 350 bp using Covaris M220 (Gene Company Limited, China) for paired-end library construction. Paired-end library was constructed using NEXTFLEX Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA). Paired-end sequencing was performed on Illumina NovaSeq (Illumina Inc., San Diego, CA, USA) using NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles). Low-quality reads (length < 50 bp or with a quality value < 20 ) were removed by fastp (version 0.23.0) and assembled into longer contigs using MEGAHIT (version 1.1.2). Contigs with a length  300 bp were selected as the final assembling result and used for further gene prediction and annotation. Open reading frames (ORFs) from each assembled contig were predicted using MetaGene. The predicted ORFs with a length  100 bp were retrieved and translated into amino acid sequences using the NCBI translation table. A non-redundant gene catalog was constructed using CD-HIT (version 4.6.1) with 90% sequence identity and 90% coverage. High-quality reads were aligned to the non-redundant gene catalogs to calculate gene abundance with 95% identity using SOAPaligner (version 2.21). Representative sequences of non-redundant gene catalog were aligned against the NR database, Kyoto Encyclopedia of Genes and Genomes database, and eggNOG database with an E-value threshold of 1e-5 to obtain species annotation information and functional annotation information on genes using Diamond (version 0.8.35).