1. SPECIFICITY and SENSITIVITY – Performance of Applied Conventional PCR Assays Supplementary Table 3: 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‘. Supplementary Table 2: Specificity and sensitivity of the 3 applied primer sets Arc, Bac and Fun for detecting Archaeal, bacterial and fungal reference organisms, were tested in conventional PCRs. Legend: -, no amplicon (correct); +, faint amplicon (correct); ++, strong amplicon (correct); +++, very strong amplicon (correct); - in gray box, no amplicon (false-positive), (+) in gray box, very faint, but visible amplicon (false-positive). NTCFungiBacteriaArchaea PrimerSetTargetA.d.AsniSaceEscoMespMeclLimoClolyClovoClopeCoprTephFlavoThleMefoMeagMethMewoMevoMeboMephMehuMemeMecoMeacMela ARC787F / ARC1059RArcArchaea------ - --- - -- - ++ (+)++ (+)++ BAC338F / BAC805RBacBacteria-(+) +++--+ +++++ ++(+) --- - - FF390 / FR1FunFungi-+++ ----------------------- 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- CONCLUSION: The applied conventional PCR assays performed highly specific in case of Archaea and fungi, while minor cross reactions could be revealed for the bacteria set. ad Material & Methods end-point PCR programs (Archaea, Bacteria, Fungi): Initial denaturation at 95°C for 8 min; 30 cycles of denaturation at 95°C for 60 sec, annealing at 43°C (Bacteria and Fungi) or 48°C (Archaea) for 60 sec, elongation at 72°C for 2 min (Archaea) or 3 min (Bacteria and Fungi), and a final elongation at 72°C for 8 min (Archaea) or 10 min (Bacteria and Fungi) respectively.