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 phi29 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. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173.

FIG. 1.

Rarefaction curves for SSU rRNA genes from amplified samples 1, 3, and 5 and native sample 5 at the 98% sequence identity level.

FIG.2.

Phylogenetic tree of SSU rRNA genes from sample 5, calculated using Jukes-Cantor corrected distances. Sequences obtained using nonamplified DNA material are indicated by filled circles, and sequences obtained from MDA-amplified DNA are indicated by open circles. For comparison, the closest relatives (known species and environmental sequences) are included in the analysis. The numbers in the circles indicate multiple independent clones with highly similar sequences (>99%) to the sequence indicated on the tree. The numbers at the nodes indicate bootstrap support (if <50, it is indicated by a small circle).

FIG.2.

Phylogenetic tree of SSU rRNA genes from sample 5, calculated using Jukes-Cantor corrected distances. Sequences obtained using nonamplified DNA material are indicated by filled circles, and sequences obtained from MDA-amplified DNA are indicated by open circles. For comparison, the closest relatives (known species and environmental sequences) are included in the analysis. The numbers in the circles indicate multiple independent clones with highly similar sequences (>99%) to the sequence indicated on the tree. The numbers at the nodes indicate bootstrap support (if <50, it is indicated by a small circle).

FIG. 3.

Phylogenetic tree of SSU rRNA genes from sample 1, calculated using Jukes-Cantor corrected distances. For comparison, the closest relatives (known species and environmental sequences) are included in the analysis. The numbers in the circles indicate multiple independent clones with highly similar sequences (>99%) to the sequence indicated on the tree. The numbers at the nodes indicate bootstrap support (if <50, it is indicated by a small circle).

FIG. 4.

Phylogenetic tree of SSU rRNA genes from sample 3, calculated using Jukes-Cantor corrected distances. For comparison, the closest relatives (known species and environmental sequences) are included in the analysis. The numbers in the circles indicate multiple independent clones with highly similar sequences (>99%) to the sequence indicated on the tree. The numbers at the nodes indicate bootstrap support (if <50, it is indicated by a small circle).