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Title:   Author  item
                                            BRUNER, EMILY - Washington State Universityitem
                                            Okubara, Patriciaitem
                                            ABI-GHANEM, RITA - Non ARS Employeeitem
                                            BROWN, DAVID - Washington State Universityitem
                                            Reardon, Catherine

                                                         - *Kate*
  Submitted to: Biotechniques
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 3/2/2015
                                    Publication  Date: 4/1/2015
                                    Citation: Bruner, E.A., Okubara, P.A., Abi-Ghanem, R., Brown, D.J., Reardon, C.L. 2015. Use of pressure cycling technology for cell lysis and recovery of bacterial and fungal communities from soil. Biotechniques. 58(4):171-180.DOI: https://doi.org/10.2144/000114273                                                              
                                Interpretive Summary: Studies of the microbial diversity of soil are challenged by the inability to collect DNA from all soil dwelling microorganisms.  Numerous methods including chemical, enzymatic, and mechanical methods are used for cell lysis and all methods are known to introduce bias to the recovery of the community DNA.  In this study, we tested whether hydrostatic pressure cycling technology (PCT) could enhance cell lysis and DNA recovery from the soil compared to two commonly used lysis methods, bead-beating and vortex disruption.  All three lysis methods successfully recovered DNA.  PCT yielded significantly less total genomic, bacterial and fungal DNA than bead-beating or vortexing; however, the PCT recovered a greater number of phylotypes or “genetic species.”  The results indicate that both measures of diversity and quantity are important when evaluating the bias of different DNA extraction methods.  Additionally, PCT may be used to recover populations unique to other extraction methods.  This study improves our understanding of methodological biases of soil DNA extraction and may facilitate comparisons between studies using different extraction methods.
                                Technical Abstract: Current molecular methodologies, specifically DNA-based approaches, provide access to previously hidden soil biodiversity and are routinely employed in environmental studies of microbial ecology. Selection of cell lysis methodology is critical to community analyses due to the inability of any singular extraction technology to recover the absolute genetic structure from environmental samples. Numerous methodologies are currently applied to interrogate soil communities, each with its own inherent bias. Improving our understanding of methodological biases will make cross comparisons more accessible. In this study, we compared the efficacy and bias of three physical cell lysis methods in conjunction with the PowerLyzer PowerSoil DNA Isolation Kit (MoBio) for direct DNA extraction from soil: bead-beating, vortex disruption, and hydrostatic pressure cycling technology (PCT). PCT lysis is novel to soil DNA extraction and was optimized for soils of varying texture prior to comparisons with traditional bead-beading and vortex disruption lysis.  All lysis methods successfully recovered DNA with the two traditional mechanical lysis techniques yielding greater genomic, bacterial, and fungal DNA per gram soil. Lysis method had a significant impact on bacterial and fungal community fingerprints. Although the abundance of bacterial and fungal genes recovered per gram soil was significantly less with PCT versus traditional mechanical lysis methods, PCT recovered a significantly greater number of phylotypes. Results indicate the importance of diversity and quantity measures when assessing DNA extraction bias as soil DNA retrieved by PCT lysis represented populations unique from the traditional mechanical lysis methods despite significantly lower DNA yields.


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