TITLE: Protocol for DNA Extraction from Urine
AUTHORS: A. Lenore Ackerman, Jennifer Tash Anger, Muhammad Umair Khalique, James E. Ackerman, Jie Tang, Jayoung Kim, David M. Underhill, Michael R. Freeman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-style:italic;">Introduction.</span><span> Recent data suggest the urinary tract hosts a microbial community of varying composition, even in the absence of infection. Culture-independent methodologies, such as next-generation sequencing of conserved ribosomal DNA sequences, provide an expansive look at these communities, identifying both common commensals and fastidious organisms. A fundamental challenge has been the isolation of DNA representative of the entire resident microbial community, including fungi. </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-style:italic;">Materials and Methods.</span><span> We evaluated multiple modifications of commonly-used DNA extraction procedures using standardized male and female urine samples, comparing resulting overall, fungal and bacterial DNA yields by quantitative PCR. After identifying protocol modifications that increased DNA yields (lyticase/lysozyme digestion, bead beating, boil/freeze cycles, proteinase K treatment, and carrier DNA use), all modifications were combined for systematic confirmation of optimal protocol conditions. This optimized protocol was tested against commercially available methodologies to compare overall and microbial DNA yields, community representation and diversity by next-generation sequencing (NGS). </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-style:italic;">Results.</span><span> Overall and fungal-specific DNA yields from standardized urine samples demonstrated that microbial abundances differed significantly among the eight methods used. Methodologies that included multiple disruption steps, including enzymatic, mechanical, and thermal disruption and proteinase digestion, particularly in combination with small volume processing and pooling steps, provided more comprehensive representation of the range of bacterial and fungal species. Concentration of larger volume urine specimens at low speed centrifugation proved highly effective, increasing resulting DNA levels and providing greater microbial representation and diversity. </span></div><style>
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							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-style:italic;">Conclusions.</span><span> Alterations in the methodology of urine storage, preparation, and DNA processing improve microbial community profiling using culture-independent sequencing methods. Our optimized protocol for DNA extraction from urine samples provided improved fungal community representation. Use of this technique resulted in equivalent representation of the bacterial populations as well, making this a useful technique for the concurrent evaluation of bacterial and fungal populations by NGS. </span></div><style>
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[STEPS]
?. Spin 30 ml for 15 minutes at 1500 X G at room temperature
?. Pour off supernatant into a separate, 50 ml tube and store at -80 c.Enzymatic Disruption
?. Resuspend pellets in 500 uL Enzyme buffer (0.5 M Tris, 1 mM EDTA, 0.2% 2-mercaptoethanol, pH 7.5) in a 1.5 ml Eppendorf tube
?. Add Enzymes:  100 uL Lyticase (2000 U/ml) and 100 uL Lysozyme (100 mg/ml)
?. Incubate at 30°C for 30 minutes, inverting every 5-10 minutes
?. Centrifuge at 4000 rcf for 5 minutes
?. Remove supernatantMechanical Disruption
?. Resuspend pellet in 800 uL Stool DNA stabilizer
?. Add Beads: 100 uL 0.1 mm beads and 300 uL 0.5 mm beads
?. Bead beat on high for 1 minute
?. Spin briefly at 17,000 X G
?. Bead beat on high for 1 minuteThermal Disruption
?. Heat at 95°C for 5 minutes
?. Vortex for 5 seconds
?. Heat at 95°C for 5 minutes
?. Vortex for 5 seconds
?. Cool on ice for 5 minutes
?. Centrifuge at 17,000 X G for 1 minuteDNA Extraction
?. For each sample, add 350 ml to each of 2 1.5 ml tubes
?. Add 10 ul proteinase K mixture (Qiagen #1017738) to each sample
?. Add 250 ul Buffer AL+Carrier [Poly (A) Sigma-Aldrich #10108626001 10ng/ml] to each sample
?. Vortex for 15 seconds
?. Incubate at 70°C for 10 minutes
?. Add 250 uL 100% EtOH to each sample
?. Mix by vortexing for 5 seconds
?. Transfer mixture from first tube to QIAamp Mini Spin Column (#1011706) and centrifuge at 17,000 X G for 1 minute
?. Change collection tube, transfer mixture from second tube to spin column  and centrifuge at 17,000 X G for 1 minute
?. Change collection tube and add 500 uL Buffer AW1 to column
?. Centrifuge at 17,000 X G for 1 minute
?. Change collection tube and add 500 uL AW2 to column
?. Centrifuge at 17,000 X G for 3 minutes
?. Remove residual EtOH by centrifuging an additional 1 min at max
?. Transfer column to new 1.5 mL Eppendorf tube
?. Add 50 uL Buffer AE to column, incubate at RT for 5 minutes and spin at 17,000 X G for 1 minute
?. Transfer fluid from collection tube back to spin column and repeat last step
?. Measure DNA concentration on Nanodrop 2000 Spectrophotometer