16S rRNA sequencing of soil microbiome
DNA was extracted from soil samples using the DNeasy PowerSoil Pro Kit (catalog number: 47014, QIAGEN, Hilden, Germany). Library preparation was performed using the NEXTflex 16S V4 Amplicon-Seq Kit 2.0 (catalog number: NOVA-4203-03, Revvity, Waltham, MA, USA). Libraries were quantified with a 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, USA), 2400 TapeStation System (catalog number: G2991BA, Agilent Technologies, Santa Clara, CA, USA) and a Qubit 4 Fluorometer (ThermoFisher Scientific, Waltham, MA USA). Samples were pooled in a NextSeq1000/2000 P1 Reagents (600 cycles) flowcell (catalog number: 20075294, Illumina, San Diego, California, USA) for 2.5 million reads per sample for 36 samples. Demultiplexed 16S rRNA sequences were imported and analyzed using DADA2 (version 1.28 (C)) in R (version 4.3.1). Trimming and filtering parameters were set as default except for truncLen=c(300,210), and maxEE=c(2,2): to keep mean base position Phred scores above 30, forward and reverse reads were trimmed to 300 and 210 base pairs, respectively, and the maximum expected errors were two per read. Default parameters were used for modeling error rates, inferring sequence variants, dereplication, and merging paired-end reads. Chimeras were removed via the consensus method and the remaining sequences were assigned taxonomies via the SILVA 138.2 reference database (Quast et al., 2013; Yilmaz et al., 2014). Sample read counts after sequencing, filtering, merging paired-end reads, and removing chimeras are available in Supplementary Table 5. Shannon and Simpson alpha diversity, Bray non-metric multidimensional scaling (NMDS), and bar plots were analyzed and generated using phyloseq (version 1.44; McMurdie et al., 2013). Analyses of variance (ANOVAs) on distance matrices were conducted using adonis2 in the vegan package (version 2.6-4).