title: A novel approach for achieving ultra-high enrichment of anaerobic ammonium oxidizing bacteria and nitrogen removal rate in municipal wastewater treatment: A pure biofilm process without suspended sludge
design: Sequencing was outsourced to Meggie Biomedical Technology Co., Ltd. (Shanghai, China), using the HISEQ4000 platform (Illumina Inc., USA) with paired-end sequencing performed using the HISEQ 3000/4000 SBS sequencing kit. SEQPREP software was used to perform quality control on the 3' and 5' ends, trimming low-quality regions from both ends of the sequences, while Sickle software was employed to filter out low-quality reads, yielding high-quality short sequence fragments. The SOAPDENOVO algorithm, based on De Bruijn graphs, was employed for genome assembly, using graph theory models and heuristic strategies to solve short-read overlap and assembly issues, and ultimately generating contigs for genome reconstruction. After assembly, MetaGene software was employed to predict open reading frames (ORFs), retrieving nucleic acid sequences longer than 100 bp from the NCBI database and identifying coding regions by translating amino acid sequences, laying the data foundation for subsequent analysis. During gene quantification analysis, Soapaligner software was used to align high-quality reads with the non-redundant gene database (NR database) to compute gene abundance values. Additionally, to classify gene functions based on sequence similarity analysis, BLASTP (version 2.2.28+) was employed to compare against the NCBI non-redundant protein database for functional annotation. Lastly, through the KEGG orthology database (KEGG Orthology), gene sequences were mapped to specific KO identifiers, and gene function annotation was performed based on the topological structure of the KEGG metabolic pathway database, pinpointing the biological role locations of the target genes within the metabolic network. All bioinformatics analyses were carried out using the Meggie Bioinformatics Cloud Platform (www.i-sanger.com).