SPECIFICITY and SENSITIVITY – Performance of Applied qPCR Assays A Bacteria Methanogenic Archaea F FBacteria F FF F Supplementary Figure 1: Specificity and sensitivity of the six applied primer sets: methanogenic Archaea (A), methanotrophic bacteria (B), Bacteroidetes (C), Bacteria (D), Firmicutes (E) and Fungi (F) for detecting corresponding reference organisms were tested in quantitative PCRs. F, fungi; for the detailed phylogenetic affiliation of the bacterial reference strains see suppl. table 1. Legend: rhombi indicate no (right-negative) amplification signals, circles indicate no (false-negative) amplification signals, dots indicate a positive (specific) amplification signal, crosses indicate a false-positive (non-specific) amplification signal.

AbbreviationMicroorganism (species)StrainClassPhylumDomainReferenceadd. relevant MefoMethanobacterium formicicum1535MethanobacteriaEuryarchaeotaArchaeaDSMZ methanogenic MeagMethanobacterium thermaggregans3266MethanobacteriaEuryarchaeotaArchaeaDSMZ MethMethanothermobacter thermautotrophicus3720MethanobacteriaEuryarchaeotaArchaeaDSMZ MewoMethanothermobacter wolfeii2970MethanobacteriaEuryarchaeotaArchaeaDSMZ MevoMethanococcus voltae1537MethanococciEuryarchaeotaArchaeaDSMZ MeboMethanoculleus bourgensis3045MethanomicrobiaEuryarchaeotaArchaeaDSMZ MephMethanoculleus thermophilus2373MethanomicrobiaEuryarchaeotaArchaeaDSMZ MehuMethanospirillum hungatei864MethanomicrobiaEuryarchaeotaArchaeaDSMZ MemeMethanomethylovorans thermophila17232MethanomicrobiaEuryarchaeotaArchaeaDSMZ MecoMethanosaeta concilii2139MethanomicrobiaEuryarchaeotaArchaeaDSMZ MeacMethanosarcina acetivorans2834MethanomicrobiaEuryarchaeotaArchaeaDSMZ MelaMethanosarcina thermophila1825MethanomicrobiaEuryarchaeotaArchaeaDSMZ EscoEscherichia coli5347γ-ProteobacteriaProteobacteriaBacteriaDSMZ- Mesp Methylosinus sporium17706 α-ProteobacteriaProteobacteria Bacteria DSMZ methanotrophic Mecl Methylomonas clara 6330γ-ProteobacteriaProteobacteria Bacteria DSMZ methanotrophic LimoListeria monocytogenes20600BacilliFirmicutesBacteriaDSMZ- ClolyClostridium cellulolyticum5812ClostridiaFirmicutesBacteriaDSMZ- ClovoClostridium cellulovorans3052ClostridiaFirmicutesBacteriaDSMZ- ClopeClostridium perfringens756ClostridiaFirmicutesBacteriaDSMZ- CoprCoprothermobacter proteolyticus5265ClostridiaFirmicutesBacteriaDSMZ- TephThermacetogenium phaeum26808ClostridiaFirmicutesBacteriaDSMZ- FlavoFlavobacterium xinjiangenseundefinedFlavobacteriiaBacteroidetesBacteriathis study- ThleThermotoga lettingae14385Thermotogae BacteriaDSMZ- SaceSaccharomyces cerevisiae70449SaccharomycetesAscomycotaFungiDSMZ- AsniAspergillus niger1957EurotiomycetesAscomycotaFungiDSMZ- Supplementary Table 1: All microbial reference strains that were applied in the specificity and sensitivity checks, were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany), except the one with the indication ‘this study‘. CONCLUSION: In general, all primer pairs detected the appropriate reference strains specifically and the detection signal was 1 to 4 orders of magnitude higher than those of non-appropriate references. Although, some primer pairs showed a distinct cross reaction potential for certain domains or groups and therefore an accurate post-analytical ascertainment and a correction of the copy numbers was necessary. The detailed specificity and sensitivity checks of the Archaea specific qPCR assay are accessible in Reitschuler et al. (2014a). Correction factors: Thereby, the detection potential of the assays for matching reference organisms was opposed to that of non-matching ones, in order to receive correction factors that were used to adjust the measured qPCR values. Standards for qPCR measurements: For the quantification and efficiency calculations we used diluted standards and plotted the C T (cycle threshold) values against the log of given templates to gain standard curves. As standards we used purified PCR products of known concentrations. Therefore we applied pure DNA from corresponding reference organisms, derived via DSMZ (German Collection of Microorganisms and Cell Cultures) or previously isolated and identified organisms, for each primer pair (Methanosarcina acetivorans [DSM No. 2834] for Arc and for MA primer, Escherichia coli [DSM No. 5347] for Bac primer, Methylobacter tundripaludum [DSM No ] for Metr primer, Flavobacterium xinjiangense [this study] for Btd primer, Clostridium cellulolyticum [DSM No. 5812] for Firm primer and Geotrichum klebahnii (Illmer et al. 2007) for Fun Primer). For more detailed specifications concerning standard preparation, calibration and efficiency calculation see Reitschuler et al. (2014a). ad Material & Methods qPCR programs (Bacteria [Bac], Bacteroidetes [Btd], Firmicutes [Firm], Fungi [Fun], methanogenic archaea [MA], methanotrophic bacteria [Metr]): Initial denaturation at 95°C for 10 min; 30 to 45 cycles of denaturation at 95°C for 25 sec [MA, Metr], 30 sec [Bac, Firm, Fun], or 60 sec [Btd]; annealing at 56°C [MA], 60 °C [Firm, Fun], 61°C [Bac], 63°C [Metr], or 64°C [Btd] for 15 sec [Bac], 25 sec [MA, Metr], 30 sec [Firm, Fun], or 60 sec [Btd]; and elongation at 72°C for 20 sec [Bac, Firm, Fun], 25 sec [Metr], or 30 sec [MA, Btd] respectively.