skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments

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 ?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 percent 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 percent of the sequences had significant similarities to known proteins, and ''clusters of orthologous groups'' (COG) analysis revealed that more than half of the sequences frommore » 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.« less

Authors:
; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
COLLABORATION - Collaboration with Diversa,SanDiego, California; Oak Ridge National Laboratory; and Virtual Institutefor Microbial Stress and Survival
Sponsoring Org.:
USDOE
OSTI Identifier:
894235
Report Number(s):
LBNL-60416
Journal ID: ISSN 0099-2240; AEMIDF; TRN: US200701%%271
DOE Contract Number:
DE-AC02-05CH11231
Resource Type:
Journal Article
Resource Relation:
Journal Name: Applied and Environmental Microbiology; Journal Volume: 72; Journal Issue: 5; Related Information: Journal Publication Date: 05/2006
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 54 ENVIRONMENTAL SCIENCES; AMPLIFICATION; CONSTRUCTION; DISTRIBUTION; DNA; DNA POLYMERASES; ESCHERICHIA COLI; GENES; NITRATES; PROTEINS; SEDIMENTS; SOILS; SPECIES DIVERSITY; STRAINS; TARGETS

Citation Formats

Abulencia, C.B., Wyborski, D.L., Garcia, J., Podar, M., Chen, W., Chang, S.H., Chang, H.W., Watson, D., Brodie,E.I., Hazen, T.C., and Keller, M. Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments. United States: N. p., 2005. Web.
Abulencia, C.B., Wyborski, D.L., Garcia, J., Podar, M., Chen, W., Chang, S.H., Chang, H.W., Watson, D., Brodie,E.I., Hazen, T.C., & Keller, M. Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments. United States.
Abulencia, C.B., Wyborski, D.L., Garcia, J., Podar, M., Chen, W., Chang, S.H., Chang, H.W., Watson, D., Brodie,E.I., Hazen, T.C., and Keller, M. Sat . "Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments". United States. doi:. https://www.osti.gov/servlets/purl/894235.
@article{osti_894235,
title = {Environmental Whole-Genome Amplification to Access Microbial Diversity in Contaminated Sediments},
author = {Abulencia, C.B. and Wyborski, D.L. and Garcia, J. and Podar, M. and Chen, W. and Chang, S.H. and Chang, H.W. and Watson, D. and Brodie,E.I. and Hazen, T.C. and Keller, M.},
abstractNote = {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 ?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 percent 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 percent 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.},
doi = {},
journal = {Applied and Environmental Microbiology},
number = 5,
volume = 72,
place = {United States},
year = {Sat Dec 10 00:00:00 EST 2005},
month = {Sat Dec 10 00:00:00 EST 2005}
}