16S rRNA gene amplicon sequencing

We assessed the methanogenic and methanotrophic community composition through 16S rRNA gene amplicon sequencing, utilizing the Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd). The detailed procedures for sequencing and operational taxonomic units (OTUs) clustering have been described in our previous study (Bao et al., 2024). OTUs were clustered at a 98.65% similarity threshold using UPARSE (version 7.1, http://drive5.com/uparse/), with chimeric sequences identified and removed via UCHIME. The phylogenetic affiliations of each 16S rRNA gene sequence were determined using the RDP Classifier (http://rdp.cme.msu.edu/) against the Silva SSU132 16S rRNA database with a confidence threshold of 70%. Taxonomic classification was further refined by aligning sequences to the Silva 16S rRNA database (v138) using an alignment threshold of 80% and the RDP classifier (Wang et al., 2007), resulting in an annotated OTU table.ReferencesBao, T., Wang, L., Huang, Y., Li, H., Qiu, L., Liu, J., Shi, L., Liu, Y., Qian, H., Ding, Y., & Jiang, Y. (2024). Elevated [CO2] reduces CH4 emissions from rice paddies under in situ straw incorporation. Agriculture, Ecosystems & Environment, 370, 109055. https://doi.org/10.1016/j.agee.2024.109055Wang, Q., Garrity, G. M., Tiedje, J. M., & Cole, J. R. (2007). Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology, 73(16), 5261-5267. https://doi.org/10.1128/AEM.00062-07