Page 233 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Fluorescence in situ hybridization (FISH) microscopy
FISH microscopy samples were taken weekly and prepared in duplicate according to the FISH protocol as described by Amann et al. (1990) using a hybridization buffer with 35% (v/v) formamide. M. sporium was targeted using bacterial EUB mix probes (5’-GCT GCC TCC CGT AGG AGT-3’; 5’-GCA GCC ACC CGT AGG TGT-3’; 5’-GCA GCC TTC CGT AGA AGT- 3’) (Daims et al. 1999). M. barkeri was targeted by a combination of ARCH-0890 probe (5’- GTG CTC CCC CGC CAA TTC CT-3’) targeting archaea (Stahl and Amann 1991) and MSMX-0860 probe (5’-GGC TCG CTT CAC CGC TTC CCT-3’) targeting Methanosarcinaceaea (Raskin et al. 1994).
Genome sequencing and data analysis of methanotrophic culture
DNA was extracted from 20 ml pelleted methanotrophic culture grown to an OD600 of ~ 1. DNA was extracted using the cetyltrimethylammoniumbromide (CTAB) extraction buffer protocol as described by Zhou et al. (1996). DNA library preparation and sequencing was performed by BaseClear on an Illumina HiSeq2500 platform using the Illumina paired end protocol (BaseClear B.V., Leiden, the Netherlands). Quality-trimming, adapter removal, and contaminant-filtering of Illumina HiSeq paired-end sequencing reads was performed using BBDUK (BBTOOLS version 37.76) (Bushnell 2014). Trimmed reads were assembled using metaSPAdes v3.11.1 (Nurk et al. 2017) at default settings. MetaSPAdes iteratively assembled the metagenome using k-mer size 21, 33, 55, 77, 99, and 127. Reads were mapped back to the metagenome using Burrows-Wheeler Aligner 0.7.17 (BWA) (Li and Durbin 2018), employing the “mem” algorithm. The sequence mapping files were handled and converted as needed using SAMtools 1.6 (Li et al. 2018). Metagenome binning was performed for contigs greater than 1,500 bp using five binning algorithms: 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 bin sets from each algorithm were supplied to DAS Tool 1.0 (Sieber et al. 2018) for consensus binning to obtain the final optimized bins. The quality of the generated bins was assessed through 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). Predicted protein sequences were submitted to
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