Page 109 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

with a 97% identity cut-off value using the 454 SOP (http://www.mothur.org/) (Schloss et al., 2009) with IonTorrent modified protocols. Chimeras were checked with the Uchime algorithm (Edgar et al. 2011), singletons were removed (Fig. S6). Taxonomy was assigned against the SILVA nr v132 database using the “mothur” taxonomy assigner (Schloss et al. 2009). Data visualization was performed using the “vegan” package in “r” (Oksanen et al. 2017). All alpha diversity indices were calculated with the OTU-based alpha diversity analysis tool summary.single() of “mothur”. Non-metric dimensional scaling (NMDS) plots were prepared in “r” using the “vegan” and “MASS” packages after pre-filtering of non-abundant OTUs (Venables and Ripley 2002). OTUs with a sum of ≤ 1 per sequencing dataset were removed from the OTU table. NMDS ordination was performed with the metaMDS() function of “vegan”. Data were processed by square root transformation and Wisconsin double standardization.
Data availability. Amplicon sequencing data were deposited in the GenBank database under the BioProject PRJNA639452
PCR quantification, cloning and qPCR. 16S rRNA gene copy numbers were quantified with the archaeal and bacterial primers described above, except that for bacterial quantification, the primer Bac806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Caporaso et al. 2012) was used. Quality and size checks were performed by agarose gel electrophoresis. All qPCR reactions were performed using PerfeCTA Quanta master mix (Quanta Bio, Beverly, MA) and 96-well optical PCR plates (Bio-Rad Laboratories B.V., Veenendaal, the Netherlands) with optical adhesive covers (Applied Biosystems, Foster City, CA). All reactions were performed on a C1000 Touch thermal cycler equipped with a CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad Laboratories B.V., Veenendaal, the Netherlands); a maximum of 1 ng of DNA template was used per reaction. Negative controls were prepared for each run by replacing the template with sterile Milli-Q water. Standard curves were constructed with a 10-fold serial dilution of a quantified copy number of pGEM®-T Easy plasmids containing inserted Illumina primer PCR fragments of the archaeal and bacterial 16S rRNA genes (Promega, Madison, WI). All qPCR data were analyzed using Bio-Rad CFX Manager version 3.0 (Bio-Rad Laboratories B.V., Veenendaal, the Netherlands).
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