Environmental Whole-Genome Amplification To Access Microbial Populations 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% 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, smallsubunit 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.Recent studies have demonstrated that natural attenuation and bioremediation of metals, radionuclides, and organic contaminants cannot be effectively applied at many sites until we have a better understanding of the physiology, ecology, and phylogeny of microbial communities at contaminated sites (12,38,57). However, the success of many monitored natural attenuation and bioremediation approaches depends largely on our understanding of regulatory mechanisms and cellular responses to different environmental factors affecting the contaminant degradation or metal reduction activity in situ. Microorganisms are often exposed to multiple stress conditions in situ, and the microbial community structure is most likely affected by many different abiotic and biotic variables in a nonlinear fashion (58).

The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses

“…We also performed complementary analyses on the unamplified DNA to confirm and quantify the trends observed in the metagenomic libraries. Recent studies reported that MDA caused relatively little biases for the sequencing of single genomes (Pinard et al, 2006), but indicated that MDA caused biases in community composition when used on mixed communities (Abulencia et al, 2006;Bodelier et al, 2009). qPCR analyses indicated here that the bias in community composition introduced by MDA is quite strong.…”

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

“…Small-insert metagenomic libraries generated from three MDA reactions shared a similar cluster of orthologous group profiles, indicating conservation of sequences by MDA. However, MDA from several microbes, experimentally mixed in known ratios, indicated that some bias may occur in their representation (Abulencia et al, 2006). In another subsurface study, Edwards et al (2006) demonstrated the first use of 454 pyrosequencing to characterize metagenomic DNA, employing MDA to obtain sufficient template using microbial assemblages from the Soudan Iron Mine in Minnesota, USA.…”

“…Abulencia et al (2006) estimated that an untenable 11-88 kg of contaminated subsurface soil (at 10 4 cells g -1 ) would be necessary to extract microgram amounts of DNA for shotgun library construction, with 20-fold more required for BAC libraries. To overcome this, MDA was successfully applied to heavily contaminated subsurface-soil samples from Oak Ridge, Tennessee, to generate sufficient template for 16S rRNA gene PCR and construction of libraries.…”

Uncultured archaea in deep marine subsurface sediments: have we caught them all?

“…Ultimately, alternative methods that do not depend on the design and employment of oligonucleotide primers may reveal to what degree cloning and sequencing of PCR products gives a representative picture of microbial biodiversity in subsurface sediments. For example, strand displacement amplification of environmental DNA (reviewed in Hutchinson et al, 2005;Zhang et al, 2006) can generate sufficient amounts of DNA for further sequence analysis from DNA-limited sediment samples (Raghunathan, 2005;Abulencia et al, 2006). The method can be combined with highly efficient pyrosequencing of deep subsurface genomic DNA (Biddle, 2006).…”