Page 85 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

≥ 99.9% nt identity). CheckM (Parks et al. 2015) confirmed presence of a single, 91.0% complete non-heterogeneous strain. Identity of the draft genome contigs was determined by performing nucleotide BLAST searches against the nt/nr collection database. The methanogen environmental draft genome bin was extracted based on a GC content between 0.475 and 0.575 and a sequencing depth between 3 and 10. The resulting bin contained 459 contigs of which 353 contigs were most identical to Methanothrix concilii GP6 (86% of bases, 2.3 Mbp, ≥ 79.9% nt identity). CheckM showed strain heterogeneity of 11.1% and completeness of 76.8%, indicating the bin likely contains a core genome of Methanothrix species. Nucleotide BLAST on nine contigs with sequencing depth > 500 indicated best hits to Geobacter metallireducens GS-15 (six sequences, ≥ 99% identity) and Azoarcus sp. (three sequences, ≥ 94% identity).
Phylogenetic 16S and 18S rRNA gene analysis
For the general species composition analysis, quality trimmed reads were mapped against the Silva SSU non-redundant database version 128 (https://www.arb-silva.de/) containing 645,151 reference sequences. Length and similarity fraction were set to 50% and 70%, respectively, and values for mismatch, insertion and deletion cost to 2, 3 and 3, resulting in a total of 28,774 mapped reads (0.59% of total). Sequences were submitted to the SILVAngs data analysis server 1.3.5 (Quast et al. 2012) and processed using default parameters. The species distribution pattern remains similar when additionally correcting for 16S rRNA gene copy numbers that are high in the Proteobacteria and Firmicutes phyla (Klappenbach et al. 2001; Acinas et al. 2004).
Reads mapping to the SSU database were extracted per genus. De novo assembly was performed using automatic word and bubble size and stringent similarity and length fraction parameters of 0.95 to obtain near-complete 16S sequences. Comparable approaches were taken previously (Bartram et al. 2011; Miller et al. 2011; Speth et al. 2016). Additionally, quality trimmed reads were mapped against the fungal internal transcribed spacer (ITS) RefSeq Targeted Loci database containing 5,362 sequences (PRJNA177353) as described above (Alvarez, Groenewald and Crous 2017).
Functional analyses
Draft genome and metagenome annotations were performed using Prokka version 1.10 with standard parameters (Seemann 2014). The Geobacter environmental draft genome was first
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