209 Methanotrophic flexibility of “Ca. Methanoperedens” and SRB in meromictic Lake Cadagno 2016) and the SILVA 16S rRNA gene reference database v138.2 (Quast et al., 2013) to assign taxonomy to amplicon sequencing variants, and also to update the taxonomy of the previously described “Desulfobulbaceae” SRB (ZOTU 307) in Su et al. (2020). Furthermore, we selected two different sediment-depths (23 cm and 25 cm) and three long-term AOM incubations (unamended control, sulfate, and manganese oxides) for downstream metagenomics (Figure 1B). Library preparation and sequencing of selected sediment and incubation DNA samples was performed at the Lausanne Genomic Technologies Facility, University of Lausanne, Switzerland (https://www.unil.ch/gtf). Paired-end sequencing (150 cycles) was done on an Illumina HiSeq 2500 using the Nextera DNA Flex protocol, generating ~ 7 Gbp/ sample for all samples except for the manganese oxides. Read quality was assessed with FASTQC v0.11.9 before and after quality trimming performed with BBDuk (BBTools v38.75). Trimmed reads were co-assembled de novo using metaSPAdes v3.14.1 (Nurk et al., 2017) and mapped to assembled contigs using BBMap (BBTools v38.75) (Bushnell, 2014). Five different assemblies were generated: (i) including sediment depths 23 cm and 25 cm reads, using the reads from the control (ii), sulfate (iii), and manganese oxides (iv) slurry incubations, and (v) a bigger coassembly including all metagenome reads (i + ii + iii+ iv) together. Contigs ≥1000bp length were used as template for read mapping. Sequence mapping files were handled and converted using Samtools v1.10., later used for binning with CONCOCT v2.1 (Alneberg et al., 2014), MaxBin2 v2.2.7 (Wu et al., 2016), and MetaBAT2 v2.12.1 (Kang et al., 2019). Generated metagenome-assembled genomes (MAGs) were dereplicated with DAS Tool v1.1.1 (Sieber et al., 2018) and taxonomically classified with the Genome Taxonomy Database Toolkit GTDB-Tk v2.1.0 (Chaumeil et al., 2019). Metagenomic mapping statistics were generated via CheckM v1.1.2 (Parks et al., 2015). For metagenomic binning, taxonomical read-recruitment assessment, and biogeography studies SingleM v0.16.0 and Sandpiper (https://sandpiper.qut.edu. au/) were used (May 2024), respectively (Woodcroft et al., 2024). MAG completeness and contamination was estimated with CheckM2 v1.0.1 (Chklovski et al., 2023). Open read frames (ORFs) in the MAGs were predicted and translated using Prodigal v2.6.3 (Hyatt et al., 2010). MAGs proteins” were annotated with DRAM 7
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