2 Metagenome Analysis
37
found in soil (Abulencia et al. 2006). MDA is also used to amplify DNA from single cells. Although this technique still has its drawbacks (Hutchison and Venter
2006) it is valuable as a method for obtaining an initial overview of the genome
of a single uncultured bacterium (Marcy et al. 2007). Such single cell approaches
aim at the isolation of single uncultured microorganisms from the environment, and
at the subsequent description of the genetic potential of each individual organism.
This approach, therefore, involves analysing the metagenome cell by cell. Single
cell genomics and metagenomics are therefore complementary approaches to assess
the genomes of uncultured organisms. Some of the techniques discussed below are
applied in both, metagenomics and single cell genomics.
2.3 Technical Challenges in Metagenome Analysis
2.3.1 Strategies to Assess the Metagenome
The construction and analysis of metagenomic libraries follows a general scheme
(Fig. 2.1). Genomic DNA is either isolated directly from an environmental sample,
or following enrichment. The DNA generally requires further purification in order to
remove contaminants such as polyphenolic substances or metal ions, which could
interfere with the subsequent enzymatic manipulation of the DNA. If insufficient
DNA is retrieved for downstream processing, multiple displacement amplification
(MDA) can be applied to increase DNA quantity (Binga et al. 2008). In the next
steps, the genomic DNA is processed for cloning into large (BAC, fosmid, Cosmid)
or small (plasmid) insert vectors or for direct DNA sequencing without prior library
construction (e.g. pyrosequencing). If libraries are constructed, arraying of individual clones is often recommended, and an initial characterisation of the library with
respect to parameters such as the number of recombinant clones, and the average
insert-size should be carried out. The libraries can be used directly for insert-end
sequencing or they can be screened for selected DNA sequences or expressed
proteins. If large insert clones carry a gene of interest, they can be completely
sequenced. This sequencing step is then followed by bioinformatic analysis of the
dataset. This includes, depending on the approach, a binning of sequences belonging to the same species or group, the assembly of contigs, the functional assignment
of predicted genes, and the interpretation of specific metabolic capabilities.
Ecologists have various microbiological and molecular tools available to them.
The choice of tool depends on the question to be answered by the metagenomic
study. Issues that need to be considered are the diversity of the microbial community
in the habitat of interest. One also needs to decide whether the study is targeted
towards the genetic capability of the entire community or of one population within
the community. Depending on this, a decision for or against an enrichment of cells
or DNA has to be made. In addition, the quality, length and amount of sequences
that will be required to address the aim of the study, has to be defined.
37
found in soil (Abulencia et al. 2006). MDA is also used to amplify DNA from single cells. Although this technique still has its drawbacks (Hutchison and Venter
2006) it is valuable as a method for obtaining an initial overview of the genome
of a single uncultured bacterium (Marcy et al. 2007). Such single cell approaches
aim at the isolation of single uncultured microorganisms from the environment, and
at the subsequent description of the genetic potential of each individual organism.
This approach, therefore, involves analysing the metagenome cell by cell. Single
cell genomics and metagenomics are therefore complementary approaches to assess
the genomes of uncultured organisms. Some of the techniques discussed below are
applied in both, metagenomics and single cell genomics.
2.3 Technical Challenges in Metagenome Analysis
2.3.1 Strategies to Assess the Metagenome
The construction and analysis of metagenomic libraries follows a general scheme
(Fig. 2.1). Genomic DNA is either isolated directly from an environmental sample,
or following enrichment. The DNA generally requires further purification in order to
remove contaminants such as polyphenolic substances or metal ions, which could
interfere with the subsequent enzymatic manipulation of the DNA. If insufficient
DNA is retrieved for downstream processing, multiple displacement amplification
(MDA) can be applied to increase DNA quantity (Binga et al. 2008). In the next
steps, the genomic DNA is processed for cloning into large (BAC, fosmid, Cosmid)
or small (plasmid) insert vectors or for direct DNA sequencing without prior library
construction (e.g. pyrosequencing). If libraries are constructed, arraying of individual clones is often recommended, and an initial characterisation of the library with
respect to parameters such as the number of recombinant clones, and the average
insert-size should be carried out. The libraries can be used directly for insert-end
sequencing or they can be screened for selected DNA sequences or expressed
proteins. If large insert clones carry a gene of interest, they can be completely
sequenced. This sequencing step is then followed by bioinformatic analysis of the
dataset. This includes, depending on the approach, a binning of sequences belonging to the same species or group, the assembly of contigs, the functional assignment
of predicted genes, and the interpretation of specific metabolic capabilities.
Ecologists have various microbiological and molecular tools available to them.
The choice of tool depends on the question to be answered by the metagenomic
study. Issues that need to be considered are the diversity of the microbial community
in the habitat of interest. One also needs to decide whether the study is targeted
towards the genetic capability of the entire community or of one population within
the community. Depending on this, a decision for or against an enrichment of cells
or DNA has to be made. In addition, the quality, length and amount of sequences
that will be required to address the aim of the study, has to be defined.