Sampling of peat soil from the acidic peatland Schlöppnerbrunnen II (Germany), DNA-stable isotope probing (DNA-SIP), total nucleic acid extraction, metagenome sequencing and assembly, and coverage-based binning were described previously (
5,
16,
29). In brief, DNA from native peat soil (10- to 20-cm depth) and DNA pooled from 16 13C-enriched fractions (density, 1.715 to 1.726 g ml−1) of a previous DNA-SIP experiment with soil from the same site (
16) was sequenced using the Illumina HiSeq 2000 system. DNA-SIP was performed after a 120-day incubation (again, 10- to 20-cm depth) that was periodically amended with small dosages of sulfate and first a mixture of unlabeled formate, acetate, propionate, and lactate for 2 weeks and thereafter a mixture of 13C-labeled formate, acetate, propionate, and lactate (all in the lower-micromolar range) (
16). Raw reads were quality filtered, trimmed, and coassembled (native soil, 385 million reads; DNA-SIP, 576 million reads) using the CLC Genomics Workbench 5.5.1 (CLC Bio). Differential coverage binning was applied to extract the
Desulfosporosinus metagenome-assembled genome (MAG) (
75). As expected (
16), the
Desulfosporosinus MAG was of low abundance in the native soil with an average coverage of 0.026 while enriched in the SIP sample with an average coverage of 34 (detailed per scaffold in
Table S1b). A side effect of sequencing a DNA-SIP sample is an apparent G+C content skew, which was normalized arbitrarily to improve binning using the following formula (
29,
76): (coverage/G+C content9) × 1015.
Scaffolds containing the 16S and 23S rRNA genes were successfully identified using paired-end linkage data (
75). Completeness, contamination, and strain heterogeneity were estimated using CheckM 1.0.6 (
77).
Phylogenomic analysis of the
Desulfosporosinus MAG was based on a concatenated set of 34 phylogenetically informative marker genes as defined by reference
77 and the Bayesian phylogeny inference method PhyloBayes using the CAT-GTR model (
78). 16S rRNA gene-based phylogeny was inferred using the ARB SILVA database r126 as a reference (
79), the SINA aligner (
80), and the substitution model testing and maximum likelihood treeing method IQ-TREE (
81). Pairwise 16S rRNA gene sequence identities were calculated with T-Coffee 11 (
82). Pairwise average nucleic and amino acid identities (ANI and AAI, respectively) (
37) between protein-encoding genes of the
Desulfosporosinus MAG and reference genomes were calculated as described previously (
29).