Before library preparation, whole-genome amplifications were performed on DNA extracts through a multiple displacement amplification step of 6 to 7 hours using the REPLI-g Midi Kit (QIAGEN) and following the manufacturer’s instructions. We monitored amplification using SYBR green I (Invitrogen) on a CFX Connect qPCR machine, stopping amplifications once the exponential phase was reached. Metagenomic libraries were prepared using the Nextera XT DNA Library Prep Kit (Illumina). Quality control and quantification of the libraries were obtained on an Agilent 2100 Bioanalyzer System using the High Sensitivity DNA reagents and DNA chips (Agilent Genomics). Metagenomic libraries were diluted to 1 nM using the Select-a-Size DNA Clean and Concentrator MagBead Kit (Zymo Research) and pooled for further sequencing on the Illumina MiniSeq platform. Contigs were assembled on CLC Genomics Workbench v. 9.5.4 (QIAGEN) using a word size of 20, a bubble size of 50, and a minimum contig length of 300 nucleotides. Reads were then mapped to the contigs using the following parameters (mismatch penalty = 3, insertion penalty = 3, deletion penalty = 3, minimum alignment length = 50% of read length, and minimum percent identity = 95%). We then performed even further stringency controls by removing any contig that had less than 5× coverage, e.g., reads per kilobase mapped (RPKM). The final resulting dataset of contigs was then used for ORF searches and BLAST analysis. Protein-encoding genes and ORFs were extracted using FragGeneScan v. 1.30 and functionally annotated against a large microbial genome database using a bioinformatics pipeline as described previously (66). Cutoff values for assigning hits to specific taxa were performed at a minimum bit score of 50, a minimum amino acid similarity of 60, and an alignment length of 50 residues.

For phylogenetic analyses, OTUs of AOAs were aligned with SINA online v.1.2.11 (67) and plotted in ARB (68) against the SILVA 16S rRNA SSU NR99 reference database release 132 (69). Closest environmental sequences with nearly full-length sequences (>1400 base pair) were selected as taxonomic references and used to calculate trees using the maximum likelihood algorithm RAxML implemented with the archaeal filter and advanced bootstrap refinement selecting the best tree among 100 replicates (70). Partial OTU sequences were added to the tree using the maximum parsimony algorithm without allowing changes of tree topology. Statistical analyses of beta-diversity were performed using R.Studio Version 3.3.0 (71) with the vegan package (72).

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