Page 60 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 3. Metal corrosion protection potential of methanogenic communities
kit (Illumina, Inc., San Diego, CA) and sequenced using an MiSeq desktop sequencer (Illumina; Macrogen, Inc., Seoul, South Korea). Samples with a low DNA yield were excluded due to technical restrictions.
(ii) Metagenome analysis. Quality-trimming, sequencing adapter removal and contaminant filtering of Illumina paired-end sequencing reads were performed using BBDuk (BBTOOLS v37.76) (Bushnell 2014). Processed reads for selected samples were coassembled de novo using metaSPAdes v3.11.1 (Nurk et al. 2017) at default settings. metaSPAdes iteratively assembled the metagenome using k-mers of length 21, 33, 55, 77, 99 and 127. Reads were mapped back to the assembled metagenome for each sample separately using Burrows-Wheeler Aligner 0.7.17 (BWA) (Li and Durbin 2018), employing the “mem” algorithm. The sequence mapping files were processed using SAMtools 1.6 (Li et al. 2018). Metagenome binning was performed for contigs greater than 1,500 bp. To optimize binning results, five binning algorithms were used: BinSanity v0.2.6.1 (Graham, Heidelberg and Tully 2017), COCACOLA (Lu et al. 2018), CONCOCT (Alneberg et al. 2014), MaxBin 2.0 2.2.4 (Wu, Simmons and Singer 2015) and MetaBAT 2 2.12.1 (Kang et al. 2015). The five bin sets were supplied to DAS Tool 1.0 (Sieber et al. 2018) for consensus binning to obtain the final bins. The quality of the generated genome bins was assessed through a single-copy marker gene analysis using CheckM 1.0.7 (Parks et al. 2015). Genomes were annotated with Prokka 1.12 (Seemann 2014) using the NCBI Reference Sequence Database (RefSeq) release 85 (Pruitt, Tatusova and Maglott 2018). The predicted coding sequences were submitted to the KEGG Automatic Annotation Server (KAAS [last update, 3 April, 2015]) (Moriya et al. 2007) for pathway analyses using the 40 most closely related organisms available in the KEGG Organisms Database as a reference (https://www.genome.jp/kegg/catalog/org_list.html; last update, 18 December 2018 [see the supplemental material for the KAAS analysis reference genomes]). Genome annotations were examined using the Artemis genome browser, release 16.0.0 (Rutherford et al. 2000). For 16S rRNA gene analysis, unprocessed Illumina MiSeq reads were mapped against the SILVA SSU nonredundant database version 132 and de novo assembled as described in in ‘t Zandt et al. (in ‘t Zandt et al. 2018). In short, sequencing reads were assembled de novo using stringent similarity and length fraction parameters of 0.95 to obtain near-complete 16S rRNA gene sequences. Specific marker protein sequences for methane cycling (methyl-coenzyme M
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