Optimization of DNA extraction from human urinary samples for mycobiome community profiling

Abstract

25Introduction. Recent data suggest the urinary tract hosts a microbial community of varying composition, 26 even in the absence of infection. Culture-independent methodologies, such as next-generation 27 sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, 28 identifying both common commensals and fastidious organisms. A fundamental challenge has been the 29 isolation of DNA representative of the entire resident microbial community, including fungi. 30 Materials and Methods. We evaluated multiple modifications of commonly-used DNA extraction 31 procedures using standardized male and female urine samples, comparing resulting overall, fungal and 32 bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA 33 yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier 34 DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. 35 This optimized protocol was tested against commercially available methodologies to compare overall and 36 microbial DNA yields, community representation and diversity by next-generation sequencing (NGS). 37 Results. Overall and fungal-specific DNA yields from standardized urine samples demonstrated that 38 microbial abundances differed significantly among the eight methods used. Methodologies that included 39 multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase 40 digestion, particularly in combination with small volume processing and pooling steps, provided more 41 comprehensive representation of the range of bacterial and fungal species. Concentration of larger 42 volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA 43 levels and providing greater microbial representation and diversity. 44 Conclusions. Alterations in the methodology of urine storage, preparation, and DNA processing improve 45 microbial community profiling using culture-independent sequencing methods. Our optimized protocol 46 for DNA extraction from urine samples provided improved fungal community representation. Use of this 49 50 51