51 Sulfide toxicity as key control on anaerobic oxidation of methane in eutrophic coastal sediments DNA extractions, amplicon sequencing and 16S rRNA gene analyses Sediments for DNA sequencing were immediately frozen at -20ºC after on board core slicing under nitrogen atmosphere and were stored at -20ºC for four months until thawing at room temperature for DNA extractions. DNA was extracted from 73 sediment samples retrieved from three cores in total, one core per site, with a depth resolution of 0.5 cm for the top 2 cm, 1 cm until 10 cm depth, 2 cm until 20 cm depth, and 4 cm below that. DNA extractions were performed with the DNeasy Power Soil Kit (Qiagen, Germany) according to the manufacturer’s instructions with a few modifications (see Supplementary Information). Amplicon sequencing was conducted by Macrogen Europe BV (Amsterdam, Netherlands) on an Illumina MiSeq platform using the archaeal primers Arch349F and Arch806R (Takai & Horikoshi, 2000), producing 2x300 bp in paired-end reads. 16S rRNA gene sequencing data was processed with the DADA2 pipeline (Lee, 2019) (see details in Supplementary Information). Metagenomic sequencing and data analyses For each site, DNA was extracted from homogenized sediment samples from four intervals: 0-4 cm, 9-12 cm, 21-24 cm, and 33-36 cm. These 12 samples were sequenced by Macrogen Europe BV (Amsterdam, Netherlands) using the TruSeq Nano DNA library with an insert size of 350 bp on an Illumina NovaSeq6000 platform, producing 2x151 bp paired-end reads. Reads were processed to generate metagenome-assembled genomes (MAGs) as previously described (Dalcin Martins et al., 2022, Chapter 4) and as detailed in the supplement. MAGs were taxonomically classified with GTDB-Tk v1 (Chaumeil et al., 2019) and annotated with DRAM v1.0 (Shaffer et al., 2020). Only high and medium quality MAGs (> 50% complete and less than 10% contaminated) were included in genome-centric analyses, and the entire dataset (binned and unbinned contigs) was considered in gene-centric analyses. For phylogenetic trees, sequences were aligned with muscle v3.8.31 (Edgar, 2004), alignment columns were stripped with trimAl v1.4.rev22 (Capella-Gutiérrez et al., 2009), and trees were built with FastTree v2.1.10 (Price et al., 2010) or UBCG v3.0 (Na et al., 2018). Average amino acid identity between selected genomes was calculated using the Konstantinidis Lab tool (http://enve-omics.ce.gatech.edu/g-matrix/index). The abundance of MAGs was inferred from normalized genome coverage (ngCOV), that is, MAG coverage normalized to total metagenome size (Dalcin Martins et al., 2
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