81 Contrasting methane, sulfide and nitrogen regimes in coastal sediment bioreactors Hilden, Germany). DNA quantities were determined by Qubit using the dsDNA HS Kit (Thermo Fisher Scientific, Waltham, MA, USA). DNA quality was determined using the NanoDrop Spectrophotometer ND-1000 (Isogen Life Science, Utrecht, The Netherlands). Our analysis included 16S rRNA gene amplicon and metagenomics raw sequencing data obtained from Dalcin Martins et al. (2024) Stockholm Archipelago sediment DNA extractions, under NCBI BioProject PRJNA805085. Raw sequencing data originated from Site 3 (Sandöfjärden) Stockholm Archipelago sediment campaign 2019 and included 16S rRNA gene amplicon -depths (in cm) 9,10,12,14,16- and metagenomic -depth- in cm: 0-4, 9-12, 21-24, and 33-36- analysis (Dalcin Martins et al., 2024, Chapter 2). 16S rRNA gene amplicon analysis Gene amplicon sequencing was performed by Macrogen Europe BV (Amsterdam, The Netherlands) on a MiSeq Illumina platform, using library kit Herculase II Fusion DNA Polymerase NEXTERA XT Index kit V2 (Illumina, Eindhoven, Netherlands), generating 2x300bp paired-end reads. Bacterial primers were Bac341F (CCTACGGGNGGCWGCAG) (Herlemann et al., 2011) and Bac806R (GGACTACHVGGGTWTCTAAT) (Caporaso et al., 2012), whereas archaeal primers were Arch349F (GYGCASCAGKCGMGAAW) and Arch806R (GGACTACVSGGGTATCTAAT) (Takai & Horikoshi, 2000). Obtained 16S rRNA gene amplicon raw sequences were processed in R Studio version v1.2.5033 and R v4.0.4 with: DADA2 package v1.18, ggplot2 v3.3.5, phyloseq v1.32, vegan v2.5.6, DESEQ2 1.28.1, dendextend v1.14, tidyr v1.1.3, viridis v0.5.1, reshape v0.8.8, zoo v1.8.8 and plyr v1.8.6. Briefly, we used the DADA2 pipeline (Callahan et al., 2016) quality plots to trim forward and reverse bacterial reads at 295bp and 260bp while archaeal reads at 295bp and 220bp, respectively. Trimming was combined with the trimLeft option 20 for primer removal. After error models were generated, sequences were dereplicated, merged, and chimeras were discarded, producing between 42.000 and 88.000 paired-end non-chimeric merged bacterial or archaeal sequences. Later, amplicon sequencing variants (ASV) were inferred and assigned using the Silva database release v.138.1 (Quast et al., 2013) downloaded from https://zenodo.org/record/4587955#.YdgiLBPMI-R. ASVs were 3
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