Genome sampling and assembly
Collection of all fluid samples and total genomic DNA extractions from those fluids, as well as corresponding physical and geochemical data have been described previously (Lau et al. , 2014, 2016; Osburnet al. , 2014; Magnabosco et al. , 2016; Heard et al. , 2017; Momper et al. , 2017). All MAGs from North America and Africa were reconstructed according to the methods used in (Momperet al. , 2017). MAG identifiers and sources are listed in Table S2. Completeness was calculated using the composite values from five widely accepted core essential gene metrics. Duplicate copies of any of these single copy marker genes was interpreted as a measure of contamination (Creevey et al. , 2011; Dupont et al. , 2012; Wu and Scott, 2012; Campbell et al. , 2013; Alneberg et al. , 2014). Individual genomes were then submitted for gene calling and annotations through the DOE Joint Genome Institute IMG-ER (Integrated Microbial Genomes expert review) pipeline (Markowitz et al. , 2008; Huntemann et al. , 2015). For quality control purposes, the genes flanking every denitrification gene presented in this study were individually searched on the NCBI RefSeq database using the BLASTp algorithm, confirming that top hits for all flanking genes were also to Chloroflexi. This step ensured that the nitrogen transforming genes of interest presented here were not simply on scaffolds that were incorrectly binned into a putative Chloroflexi genome.