DNA extraction and high-throughput sequencing

Approximately 1 g of pit mud from each sample was used for DNA extraction. The total genomic DNA of the pit mud microbial community was extracted using a Power Soil DNA Extraction Kit (QIAGEN, Germany). DNA concentration was determined using a Nanodrop 2000 spectrophotometer and diluted to 10 ng/µL for polymerase chain reaction amplification. The V4–V5 hypervariable region of the prokaryotic 16S rRNA gene isolated from the samples was amplified using prokaryotic universal primers 515F (5′-GTGYCAGCMGCCGCGGTA-3′) and 909R (5′-CCCCGYCAATTCMTTTRAGT-3′) (Tamaki et al., 2011). The 5′ end of primer 515F was connected to a 12 nucleotide barcode sample-specific sequence for subsequent sample division. Sequencing was conducted using the Illumina HiSeq platform at Guangdong Meilikang Bio-Science Ltd., China (Ni et al., 2021).

The raw reads were merged using FLASH 1.2.8 and filtered using QIIME 1.9.0 (Caporaso et al., 2010) to remove sequences with one or more of the following attributes: mismatch with primers, length of less than 300 bp, presence of ambiguous base “N,” or average base quality of less than 30 (Ni et al., 2017). Chimeric sequences were detected and removed from the remaining sequences using UCHIME (Edgar et al., 2011). The remaining high-quality sequences were then clustered into operational taxonomic units (OTUs) based on 97% sequence similarity using USEARCH (Edgar, 2013). Alpha diversity indexes and weighted UniFrac distances were calculated, and principal coordinates analysis (PCoA) based on weighted UniFrac distances was conducted using QIIME 1.9.0. The OTUs were annotated to taxa using the Ribosomal Database Project Classifier (Wang et al., 2007) with the Greengenes gg_13_8 dataset (http://qiime.org/home_static/dataFiles.html).