title: 16S rRNA Sludge Metagenome
design: Total DNA extractions from sludge samples were performed in triplicate using DNeasy PowerSoil Kit (QIAGEN, Gilden, Germany) following the manufacturers protocol. Triplicate-extracted DNA from each sample was mixed to minimize the potential variation during the DNA extraction process. All the extracted DNAs were stored at  20 C for subsequent Illumina sequencing. The V3-V4 hypervariable region of the 16S rRNA gene targeting Bacteria and Archaea was amplified using the primers 341F, 5-CCTACG GGA GGC AGC AG-3 and 806R, 5- GGA CTA CHV GGG TWT CTA AT -3 (Zhang et al., 2017). The amplification of the 16S rRNA gene was performed with a final volume of 30 L containing 15 L of PhusionR High-Fidelity PCR Master Mix (New England Biolabs), 0.2 mM of each primer, and 10 ng of template DNA. Amplicons from different samples were purified using PCR purification kit (Solgent, Korea). Amplified DNA was sequenced using the Illumina MiSeq platform (Macrogen, Seoul, South Korea) in 301-bp paired-end format at Macrogen. The raw sequences were demultiplexed to assign to samples, and removed barcode and primer sequence were using the Cutadapt tool. The raw reads were processed using DADA2 with the standard processing steps, quality filtering, denoising, merging, ASV inference, and chimeric sequence removal to obtain the amplicon sequence variants (ASVs). The amplicon sequences variants (ASVs) were also resolved by removing chimeric sequences with a reference database (Silva database) based on the "consensus" method. Subsequent analysis of the richness indices (Chao 1 estimates), diversity indices (Shannon index), and Good's coverage were performed. In addition, QIIME2 was used to evaluate the similarities of bacterial communities between the sludge samples using the unweighted pair group method with arithmetic mean (UPGMA) clustering based on a weighted UniFrac distance matrix. The taxonomic assignment was determined using BLAST against the NCBI 16S microbial database.