Part C | 17.2
460 Part C Marine Genomics
of the environmental DNA (deoxyribonucleic acid) enables the renovation of metabolic and functional traits.
This approach is generally termed environmental genomics or metagenomics. It has been effectively applied
on several environmental samples, including marine
sponges.
17.2 The Principle of Metagenomics and Related Techniques
Microbial research radically changed after 1985 due
to the pioneering work on rRNA genes by Carl
Woese [17.23]. This was followed by the work of
Pace and colleagues, who directly analyzed the 5S and
16S rRNA genes from environmental samples without
culturing [17.24]. The next technical step was the development of PCR and primers lead to amplifying the
entire gene. This advancement in technology provided
a larger microbial diversity that was not distorted by
culturing bias.
Each organism in an environment has a unique set
of genes in its genome; the combined genomes of all
the community members make up the metagenome.
Metagenome technology (metagenomics) has led to
the accumulation of DNA sequences, and these sequences are exploited for novel biotechnological applications [17.25]. Metagenomics and the associated
strategies have arrived at the forefront of biology, primarily because of the exploitation of next generation
DNA sequencing technologies. This has significantly
improved the potential for sequencing large environmental sets. Technological advances have created new
opportunities for large-scale sequencing projects that
would have been difficult to imagine several years
ago. The next key development is an emerging appreciation for the importance of complex microbial
communities in the marine environment. Due to the
overwhelming majority of unculturable microbes in
the marine environment, metagenome searches will always result in the identification of unknown genes
and proteins. Currently, single-cell genomics is well
suited for symbiosis research including bacterial symbionts of marine sponges. Thus, the probability of
uncovering unknown sequences makes this approach
more favorable than searches in cultured microbes
(Fig. 17.2).
17.2.1 Isolation of Metagenomic DNA
Metagenomics has been confirmed to be a potent tool
for studying the diverse environmental microorganism
resources. Currently, there are two approaches in research on sponge metagenomics. In the first approach,
a massive sequencing facility is employed to decode the
metagenomic library with short sequences of fragments
for assembly [17.26]. In the second one, scientists focus
on the potential of functional expression of metagenomic library clones with large insert DNA [17.27]. In
all cases, it starts with isolating DNA from the sponge
sample.
Several methods are available for extracting DNA
from sponge samples. Commercial kits are also available for extraction and purification of metagenomic
DNA, which are easy to use with significant reproducibility. Isolation of metagenomic DNA from sponge
samples can be classified into direct, indirect, and enrichment methods. The ultimate methodology should
include cell lysis and DNA extraction. The direct
method is based on cell lysis within the sample matrix
and subsequent separation of DNA from the matrix and
cell debris. Indirect DNA isolation is based on the separation of cells from the matrix, followed by cell lysis
and DNA extraction. The enrichment method involves
the separation of cells from the matrix, followed by cell
enrichment, lysis, and DNA extraction [17.28].
17.2.2 Library Construction
Sponge metagenomic DNA is composed of a promising source of novel metabolites. The discovery of these
novel metabolites requires the construction of a metagenomic DNA library and its efficient screening. During
the construction of the metagenomic library two parameters have been widely considered. The first one is the
large size and this should be the mixture of all bacterial genomes colonizing in a given environment. Such
diversity improves cloning efficiency, so that the gene
libraries provide sufficient functional genes of the entire metagenome. The second parameter is the size and
cluster organization of genes involved in the secondary
metabolite synthesis. Since the genes encoding the secondary metabolite biosynthesis are normally clustered,
clones in the metagenomic libraries should contain
larger DNA. Isolation of high molecular weight DNA
facilitates the cloning of DNA into bacterial artificial
chromosomes (BACs) and allows the characterization
460 Part C Marine Genomics
of the environmental DNA (deoxyribonucleic acid) enables the renovation of metabolic and functional traits.
This approach is generally termed environmental genomics or metagenomics. It has been effectively applied
on several environmental samples, including marine
sponges.
17.2 The Principle of Metagenomics and Related Techniques
Microbial research radically changed after 1985 due
to the pioneering work on rRNA genes by Carl
Woese [17.23]. This was followed by the work of
Pace and colleagues, who directly analyzed the 5S and
16S rRNA genes from environmental samples without
culturing [17.24]. The next technical step was the development of PCR and primers lead to amplifying the
entire gene. This advancement in technology provided
a larger microbial diversity that was not distorted by
culturing bias.
Each organism in an environment has a unique set
of genes in its genome; the combined genomes of all
the community members make up the metagenome.
Metagenome technology (metagenomics) has led to
the accumulation of DNA sequences, and these sequences are exploited for novel biotechnological applications [17.25]. Metagenomics and the associated
strategies have arrived at the forefront of biology, primarily because of the exploitation of next generation
DNA sequencing technologies. This has significantly
improved the potential for sequencing large environmental sets. Technological advances have created new
opportunities for large-scale sequencing projects that
would have been difficult to imagine several years
ago. The next key development is an emerging appreciation for the importance of complex microbial
communities in the marine environment. Due to the
overwhelming majority of unculturable microbes in
the marine environment, metagenome searches will always result in the identification of unknown genes
and proteins. Currently, single-cell genomics is well
suited for symbiosis research including bacterial symbionts of marine sponges. Thus, the probability of
uncovering unknown sequences makes this approach
more favorable than searches in cultured microbes
(Fig. 17.2).
17.2.1 Isolation of Metagenomic DNA
Metagenomics has been confirmed to be a potent tool
for studying the diverse environmental microorganism
resources. Currently, there are two approaches in research on sponge metagenomics. In the first approach,
a massive sequencing facility is employed to decode the
metagenomic library with short sequences of fragments
for assembly [17.26]. In the second one, scientists focus
on the potential of functional expression of metagenomic library clones with large insert DNA [17.27]. In
all cases, it starts with isolating DNA from the sponge
sample.
Several methods are available for extracting DNA
from sponge samples. Commercial kits are also available for extraction and purification of metagenomic
DNA, which are easy to use with significant reproducibility. Isolation of metagenomic DNA from sponge
samples can be classified into direct, indirect, and enrichment methods. The ultimate methodology should
include cell lysis and DNA extraction. The direct
method is based on cell lysis within the sample matrix
and subsequent separation of DNA from the matrix and
cell debris. Indirect DNA isolation is based on the separation of cells from the matrix, followed by cell lysis
and DNA extraction. The enrichment method involves
the separation of cells from the matrix, followed by cell
enrichment, lysis, and DNA extraction [17.28].
17.2.2 Library Construction
Sponge metagenomic DNA is composed of a promising source of novel metabolites. The discovery of these
novel metabolites requires the construction of a metagenomic DNA library and its efficient screening. During
the construction of the metagenomic library two parameters have been widely considered. The first one is the
large size and this should be the mixture of all bacterial genomes colonizing in a given environment. Such
diversity improves cloning efficiency, so that the gene
libraries provide sufficient functional genes of the entire metagenome. The second parameter is the size and
cluster organization of genes involved in the secondary
metabolite synthesis. Since the genes encoding the secondary metabolite biosynthesis are normally clustered,
clones in the metagenomic libraries should contain
larger DNA. Isolation of high molecular weight DNA
facilitates the cloning of DNA into bacterial artificial
chromosomes (BACs) and allows the characterization