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Optimization of DNA extraction from human urinary samples for mycobiome community profiling PLOS ONE Ackerman, A., Anger, J., Khalique, M., Ackerman, J. E., Tang, J., Kim, J., Underhill, D. M., Freeman, M. R., Clemens, J., Hanno, P., Kirkali, Z., Kusek, J. W., Landis, J., Lucia, M., Moldwin, R. M., Mullins, C., Pontari, M. A., Lucia, M., van Bokhoven, A., Osypuk, A. A., Dayton, R., Triolo, C. S., Jonscher, K. R., Sullivan, H. T., Wilson, R., Grasmick, Z. D., Mullins, C., Kusek, J. W., Kirkali, Z., Bavendam, T. G., Landis, J., Barrell, T., Doe, R., Farrar, J. T., Fernando, M., Gallagher, L., Hanno, P., Hou, X., Howard, T., Jemielita, T., Kuzla, N., Moldwin, R. M., Newcomb, C., Pontari, M. A., Robinson-Garvin, N., Smith, S., Stephens-Shields, A., Wang, Y., Wang, X., Klumpp, D. J., Schaeffer, A. J., Apkarian, A., Arroyo, C., Bass, M., Cella, D., Farmer, M. A., Fitzgerald, C., Gershon, R., Griffith, J. W., Heckman, C. J., Jiang, M., Keefer, L., Lloyd, R., Marko, D. S., Michniewicz, J., Miller, R., Parrish, T., Tu, F., Yaggi, R., Mayer, E. A., Rodriguez, L. V., Alger, J., Ashe-McNalley, C. P., Ellingson, B., Heendeniya, N., Kilpatrick, L., Cara, K., Kutch, J., Labus, J. S., Naliboff, B. D., Randal, F., Smith, S. R., Kreder, K. J., Bradley, C. S., Eno, M., Greiner, K., Luo, Y., Lutgendorf, S. K., O'Donnell, M. A., Ziegler, B., Schrepf, A., Hardy, I., Magnotta, V., Clauw, D. J., Clemens, J., As-Sanie, S., Berry, S., Grayhack, C., Halvorson, M. E., Harris, R., Harte, S., Ichesco, E., Oldendorf, A., Scott, K. A., Williams, D. A., Buchwald, D., Afari, N., Bacus, T., Edwards, T., Krieger, J., Maravilla, K., Miller, J., Patrick, D., Qin, X., Richey, S., Risques, R., Robertson, K., Ross, S. O., Spiro, R., Strachan, E., Sundsvold, T. J., Sutherland, S., Yang, C. C., Andriole, G. L., Lai, H., Bristol, R. L., Gereau, R. W., Hong, B. A., Klim, A. P., Sutcliffe, S., Vetter, J., Song, D. G., Milbrandt, M., Haroutounian, S., Vijairania, P., Parker (Chaturvedi), K., Tran Hung, Colditz, G., Gardner, V. C., Henderson, J. P., Spitznagle, T. M., Pakpahan, R., James, A., Yan, Y., Langston, M., Hong, B., Mueller, S., Crowley, J., Vogt, S., Hultgren, S., Nang Nguyen, Blasche, G., Qiu, C., Cupps, L., Bok, S., Hooten, T. M., Grullon, L., Atis, N., Ness, T. J., Deutsch, G., Den Hollander, J., Corbitt, B. D., Bradley, L., North, C. S., Downs, D., Anger, J., Ackerman, J., Ackerman, A., Cha, J., Eilber, K., Freeman, M., Funari, V., Kim, J., Van Eyk, J., Yang, W., Moses, M. A., Briscoe, A. C., Briscoe, D., Curatolo, A., Froehlich, J., Lee, R. S., Sachdev, M., Solomon, K. R., Steen, H., Mackey, S., Bagarinao, E., Foster, L. C., Hubbard, E., Johnson, K. A., Martucci, K. T., Mccue, R. L., Moericke, R. R., Nilakantan, A., Noor, N., Nickel, J., Ehrlich, G. D., NIH Multidisciplinary Approach Stu 2019; 14 (4): e0210306

Abstract

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.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).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.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.

View details for DOI 10.1371/journal.pone.0210306

View details for Web of Science ID 000465519100004

View details for PubMedID 31022216

View details for PubMedCentralID PMC6483181