SPECIFICITY and SENSITIVITY – Performance of Applied qPCR Assays

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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.

Abbreviation Microorganism (species) Strain Class Phylum Domain Reference add. relevantMefo Methanobacterium formicicum 1535 Methanobacteria Euryarchaeota Archaea DSMZ

methanogenic

Meag Methanobacterium thermaggregans 3266 Methanobacteria Euryarchaeota Archaea DSMZMeth Methanothermobacter thermautotrophicus 3720 Methanobacteria Euryarchaeota Archaea DSMZMewo Methanothermobacter wolfeii 2970 Methanobacteria Euryarchaeota Archaea DSMZMevo Methanococcus voltae 1537 Methanococci Euryarchaeota Archaea DSMZMebo Methanoculleus bourgensis 3045 Methanomicrobia Euryarchaeota Archaea DSMZMeph Methanoculleus thermophilus 2373 Methanomicrobia Euryarchaeota Archaea DSMZMehu Methanospirillum hungatei 864 Methanomicrobia Euryarchaeota Archaea DSMZMeme Methanomethylovorans thermophila 17232 Methanomicrobia Euryarchaeota Archaea DSMZMeco Methanosaeta concilii 2139 Methanomicrobia Euryarchaeota Archaea DSMZMeac Methanosarcina acetivorans 2834 Methanomicrobia Euryarchaeota Archaea DSMZMela Methanosarcina thermophila 1825 Methanomicrobia Euryarchaeota Archaea DSMZEsco Escherichia coli 5347 γ-Proteobacteria Proteobacteria Bacteria DSMZ -Mesp Methylosinus sporium 17706 α-Proteobacteria Proteobacteria Bacteria DSMZ methanotrophicMecl Methylomonas clara 6330 γ-Proteobacteria Proteobacteria Bacteria DSMZ methanotrophicLimo Listeria monocytogenes 20600 Bacilli Firmicutes Bacteria DSMZ -Cloly Clostridium cellulolyticum 5812 Clostridia Firmicutes Bacteria DSMZ -Clovo Clostridium cellulovorans 3052 Clostridia Firmicutes Bacteria DSMZ -Clope Clostridium perfringens 756 Clostridia Firmicutes Bacteria DSMZ -Copr Coprothermobacter proteolyticus 5265 Clostridia Firmicutes Bacteria DSMZ -Teph Thermacetogenium phaeum 26808 Clostridia Firmicutes Bacteria DSMZ -Flavo Flavobacterium xinjiangense undefined Flavobacteriia Bacteroidetes Bacteria this study -Thle Thermotoga lettingae 14385 Thermotogae Thermotogae Bacteria DSMZ -Sace Saccharomyces cerevisiae 70449 Saccharomycetes Ascomycota Fungi DSMZ -Asni Aspergillus niger 1957 Eurotiomycetes Ascomycota Fungi DSMZ -

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 CT

(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. 17260] 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.