Page 84 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 4. Robustness of coal microbial community after nutrient amendment
were checked with the Uchime algorithm (Edgar et al. 2011). Taxonomy was assigned against the SILVA nr v123 database using the MOTHUR taxonomy assigner (Schloss et al. 2009). Data visualization was performed using the “vegan” package in R (Oksanen et al. 2017).
Metagenome sequencing of the 25-month DNA sample was performed on the IonTorrent PGMTM. All library preparation and sequencing steps were performed according to the manufacturer’s instructions (Ion PGMTM Template OT2 400 Kit, Life Technologies, Carlsbad CA, United States). 100 ng genomic DNA was sheared in four one-minute shear one-minute cool down cycles using a Bioruptor® Sonicator (Diagenode, Denville NJ, United States). The library was prepared using the Ion Plus Fragment Library Kit protocol. Size distribution and concentration of the library was determined using the Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara CA, United States). Sequencing was performed using the Ion 318 Chip Kit v2.
Raw reads were quality checked using the CLC Genomics Workbench 8.0 (CLCbio, Aarhus, Denmark). Ambiguous sequences were trimmed off using an ambiguous limit of 2 and a trim limit of 0.5. On both the 3’ and 5’ side the three terminal nucleotides were discarded. To reduce noise, reads were subsequently filtered, targeting reads with a length between 30 and 400 nt (5,153,485 reads) for de novo assembly and 100-400 nt (4,884,119 reads) for 16S rRNA and functional gene analysis. For de novo assembly, minimum contig size was set to 1,000, word size to 35 and bubble size to 5,000. Values for mismatch, insertion and deletion cost were set to 2, 3 and 3, respectively, resulting in a total of 12,527 contigs with an average length of 2,624 base pairs. 85% of the reads (1.1 Gbp) were assigned to a specific contig based on a length and similarity fraction of 50% and 80%, respectively.
Analysis of environmental genomes
The assembled contig data set was analyzed based on average read coverage and GC content per contig using R (https://www.r-project.org/) (R Core Team 2014) and RStudio v3 (https://www.rstudio.com/) (RStudio Team 2014) with packages “ggplot2” (Wickham 2009) and “RColorBrewer 1.1-2” (Neuwirth 2011) (https://cran.r-project.org/web/packages/). The Geobacter environmental draft genome bin was extracted based on a GC content between 0.525 and 0.65 and a sequencing depth between 100 and 325. The resulting bin contained 139 contigs of which 118 were most identical to Geobacter metallireducens GS-15 (97% of bases, 3.4 Mbp,
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