Negative-control-anchored urinary microbiome profiling with absolute 16S quantificationAccetto, Tomaž (Avtor) Strašek Smrdel, Katja (Avtor) Taskovska, Milena (Avtor) Starčič Erjavec, Marjanca (Avtor) Smrkolj, Tomaž (Avtor) Seme, Katja (Avtor) Erdani-Kreft, Mateja (Avtor) bladder cancermidstream urinemicrobiotaurinary tract infectionnext-generation sequencingnonmetric multidimensional scalingabsolute quantificationnegative controlslow biomassRecent studies utilizing 16S rRNA amplicon sequencing have challenged the notion of urine sterility, yet urine is a low-biomass specimen in which apparent community profiles can be strongly influenced by background signal from reagents and processing. To address this interpretability gap, we integrate culture-independent absolute 16S rRNA gene quantification with urinary 16S amplicon sequencing in a negative-control-anchored workflow. Bacterial load provides a biomass-aware quality control gate that defines interpretable low-biomass thresholds and objective exclusion criteria. As a pilot application, we compared midstream urine collected prior to instrumentation from healthy volunteers and newly diagnosed bladder cancer (BC) patients, quality filtering retained 29 controls and 5 BC cases. Samples > 106 copies/ml typically produced > 10 000 reads; near 105 copies/mlread counts dropped sharply yetremained distinguishable from background. Thirteen negative controls (V3–V4 PCR and stabilization buffer; median 90, mean 124 reads) supported excluding samples with < 1000 reads. Median bacterial load was lower in BC than in controls (7.0 × 103 vs 1.07 × 106 copies/ml), although not significant in this underpowered cohort (P = 0.07). This cohort-size-independent framework enables load-based triage for sequencing, reduces backgrounddriven over-interpretation in low-biomass urine datasets, and supports modeling bacterial load as a covariate or stratifier in future studies of the bladder cancer microbiome.20262026-07-30 12:41:54Članek v reviji185282UDK: 579:616.6ISSN pri članku: 0378-1097DOI: 10.1093/femsle/fnag020COBISS_ID: 272226819sl