Page 159 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

primers were Arch349F (5’-GYGCASCAGKCGMGAAW-3’) and Arch806R (5’- GGACTACVSGGGTATCTAAT-3’) (Takai and Horikoshi 2000). For the output data, 90% of the reads had a Q20 quality score of 90% (Q30 for ≥ 81% of reads) or higher and average total read count was 161,000 reads per sample. Sequences were filtered for a length between 400 and 500 base pairs (bp). For additional analyses with single-end reads minimum read length was set to 291 bp. Amplicon sequences were quality checked for chimeras using the UCHIME algorithm (Edgar et al. 2011), clustered into OTUs with a 97% identity cut-off value and classified using the SILVA v128 16S rRNA gene non-redundant database (SSURef_NR99_128_SILVA) and the Bayesian classifier (“wang”) using the MySeq SOP (http://www.mothur.org/) (Schloss et al. 2009). “Chloroplasts”, “Mitochondria”, “unknown” and “Eukaryota” were removed for both datasets, “Bacteria” and “Archaea” were removed for the archaeal and bacterial sequence datasets, respectively. The “filename.taxonomy” and “filename.shared” files were used for downstream analyses as described in in ‘t Zandt et al. (2018). Singletons were removed and data was rarefied in “R” (see rarefraction curves in Figure S6-S9). All sequencing data were submitted to the GenBank databases under the BioProject SRP133831. Diversity indices were calculated on the OTU tables with the “summary.single” command in “mothur” using “chao” to assess community richness; “simpsoneven” and “shannoneven” to assess community evenness and “simpson” and “Shannon” to assess community diversity. NMDS plots were made using the OTU output tables from “mothur”. All data was analyzed using “R” (https://www.r-project.org/) (R Core Team 2014) and Rstudio v3 (https://www.rstudio.com/) (RStudio Team 2014). All OTUs with 1 hit or less per sample were removed to reduce noise. Plots were made using the “metaMDS” tool from the “vegan” package in “R” (Oksanen et al. 2018), stress was calculated using 20 iterations. Color labels were added with “rgl” (Adler et al. 2018) .
Cloning, sequencing and qPCR. 16S rRNA gene copy numbers in the environmental samples and enrichment samples were quantified with the archeal and bacterial Illumina primers described above. Quality and size checks were performed using agarose gel electrophoresis. All qPCR reactions were performed using PerfeCTA Quanta master mix (Quanta Bio, Beverly MA, USA) and 96-well optical PCR plates (Bio-Rad Laboratories B.V., Veenendaal, the Netherlands) with optical adhesive covers (Applied Biosystems, Foster City CA, USA). All
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