Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments

@article{citeulike:2678872, abstract = {Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using {phi}29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2\% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9\% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. 10.1128/AEM.72.5.3291-3301.2006}, author = {Abulencia, Carl B. and Wyborski, Denise L. and Garcia, Joseph A. and Podar, Mircea and Chen, Wenqiong and Chang, Sherman H. and Chang, Hwai W. and Watson, David and Brodie, Eoin L. and Hazen, Terry C. and Keller, Martin }, citeulike-article-id = {2678872}, doi = {10.1128/AEM.72.5.3291-3301.2006}, journal = {Appl. Environ. Microbiol.}, keywords = {mda, phi29, shotgun\_cloning}, month = {May}, number = {5}, pages = {3291--3301}, posted-at = {2008-05-08 23:34:52}, priority = {2}, title = {Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments}, url = {http://dx.doi.org/10.1128/AEM.72.5.3291-3301.2006}, volume = {72}, year = {2006} }

TY - JOUR ID - citeulike:2678872 L3 - citeulike-article-id:2678872 TI - Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments JF - Appl. Environ. Microbiol. VL - 72 IS - 5 SP - 3291 EP - 3301 N2 - Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using {phi}29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. 10.1128/AEM.72.5.3291-3301.2006 KW - mda KW - phi29 KW - shotgun_cloning AU - Abulencia, Carl AU - Wyborski, Denise AU - Garcia, Joseph AU - Podar, Mircea AU - Chen, Wenqiong AU - Chang, Sherman AU - Chang, Hwai AU - Watson, David AU - Brodie, Eoin AU - Hazen, Terry AU - Keller, Martin PY - 2006/05/01/ UR - http://dx.doi.org/10.1128/AEM.72.5.3291-3301.2006 ER -