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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}
}
  • 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 biasmore » 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.« less
  • No abstract prepared.
  • Historically, cholera outbreaks have been linked to V. cholerae O1 serogroup strains or its derivatives of the O37 and O139 serogroups. A genomic study on the 2010 Haiti cholera outbreak strains highlighted the putative role of non O1/non-O139 V. cholerae in causing cholera and the lack of genomic sequences of such strains from around the world. Here we address these gaps by scanning a global collection of V. cholerae strains as a first step towards understanding the population genetic diversity and epidemic potential of non O1/non-O139 strains. Whole Genome Mapping (Optical Mapping) based bar coding produces a high resolution, orderedmore » restriction map, depicting a complete view of the unique chromosomal architecture of an organism. To assess the genomic diversity of non-O1/non-O139 V. cholerae, we applied a Whole Genome Mapping strategy on a well-defined and geographically and temporally diverse strain collection, the Sakazaki serogroup type strains. Whole Genome Map data on 91 of the 206 serogroup type strains support the hypothesis that V. cholerae has an unprecedented genetic and genomic structural diversity. Interestingly, we discovered chromosomal fusions in two unusual strains that possess a single chromosome instead of the two chromosomes usually found in V. cholerae. We also found pervasive chromosomal rearrangements such as duplications and indels in many strains. The majority of Vibrio genome sequences currently in public databases are unfinished draft sequences. The Whole Genome Mapping approach presented here enables rapid screening of large strain collections to capture genomic complexities that would not have been otherwise revealed by unfinished draft genome sequencing and thus aids in assembling and finishing draft sequences of complex genomes. Furthermore, Whole Genome Mapping allows for prediction of novel V. cholerae non-O1/non-O139 strains that may have the potential to cause future cholera outbreaks.« less
  • Historically, cholera outbreaks have been linked to V. cholerae O1 serogroup strains or its derivatives of the O37 and O139 serogroups. A genomic study on the 2010 Haiti cholera outbreak strains highlighted the putative role of non O1/non-O139 V. cholerae in causing cholera and the lack of genomic sequences of such strains from around the world. Here we address these gaps by scanning a global collection of V. cholerae strains as a first step towards understanding the population genetic diversity and epidemic potential of non O1/non-O139 strains. Whole Genome Mapping (Optical Mapping) based bar coding produces a high resolution, orderedmore » restriction map, depicting a complete view of the unique chromosomal architecture of an organism. To assess the genomic diversity of non-O1/non-O139 V. cholerae we applied a Whole Genome Mapping strategy on a well-defined and geographically and temporally diverse strain collection, the Sakazaki serogroup type strains. Whole Genome Map data on 91 of the 206 serogroup type strains support the hypothesis that V. cholerae has an unprecedented genetic and genomic structural diversity. Interestingly, we discovered chromosomal fusions in two unusual strains that possess a single chromosome instead of the two chromosomes usually found in V. cholerae. We also found pervasive chromosomal rearrangements such as duplications and indels in many strains. The majority of Vibrio genome sequences currently in public databases are unfinished draft sequences. The Whole Genome Mapping approach presented here enables rapid screening of large strain collections to capture genomic complexities that would not have been otherwise revealed by unfinished draft genome sequencing and thus aids in assembling and finishing draft sequences of complex genomes. Furthermore, Whole Genome Mapping allows for prediction of novel V. cholerae non-O1/non-O139 strains that may have the potential to cause future cholera outbreaks.« less