1 Genomics in the Discovery and Monitoring
11
diversity has now been attempted with a variety of techniques including functional
and environmental genomics, transcriptomics (using microarrays and quantitative
PCR), proteomics and metabolic/metabolomic studies. Such studies have provided
crucial clues to the functional significance of natural biodiversity through the study
of well-known metabolic pathways.
Initially, perhaps the greatest success in marine functional genomics was
achieved using bacterial artificial chromosome (BAC) and fosmid (f1 origin-based
cosmid vector) libraries that allowed up to 300 kb of DNA inserts to be studied in
E. coli. A multiplex PCR screen for rRNA fragments allows the identification of
clones from specific phylogenetic groups. Full sequencing can then allow metabolic
genes to be identified in order to infer the metabolic potential of the specific organism or phylogenetic group. Such an advance was first achieved by Stein et al.
1996 with a marine Archaea population from coastal waters, followed by (Suzuki
et al. 2004)’s picoplankton study. BAC libraries derived from marine environments are now commonplace, yet still often require hundreds, if not thousands
of litres of seawater to produce. Recent modifications include the use of terminal restriction fragment length polymorphism (TRFLP) and internal transcribed
space, length heterogeneity PCR (ITS-LH-PCR) to screen for rRNA genes (Suzuki
et al. 2004, Babcock et al. 2007). Perhaps the greatest success story to come
from the BAC approach is the identification of a novel light driven photon pump
(now known as a bacteriorhodopsin) in an uncultured SAR86 (γ proteobacterial)
group BAC fragment (Beja et al. 2000). Since then the BAC approach has been
used with both natural marine communities and specific marine strains such as the
gammaproteobacterium Congregibacter litoralis (Fuchs et al. 2007), Eastern and
Pacific oysters Crassostrea virginica and C. gigas (Cunningham et al. 2006) and
protochordate Botryllus schlosseri (de Tomaso and Weissman 2003).
In addition to large-scale conventional sequencing approaches, the development
of pyrosequencing technologies now allows direct shotgun sequencing of environmental samples (Blow 2008) on a hitherto unforeseen scale (Huse et al. 2007,
Huber et al. 2007, Mou et al. 2008). Such metagenomic approaches (Handelsmann
2004) have had major implications for the marine environment with its massive
pool (quite literally) of unstudied and unculturable organisms. To date, combinations of chain-termination and pyrosequencing approaches have been used to study
marine bacterial, eukaryotic and viral diversity, sampled from diverse marine environments (coastal, open ocean, surface, deep sea, coral, subseafloor) (Breitbart et al.
2004, Venter et al. 2004, Angly et al. 2006, Culley et al. 2006, Sogin et al. 2006,
Bench et al. 2007, Biddle et al. 2008, Dinsdale et al. 2008, Quaiser et al. 2008,
Williamson et al. 2008). It is important to note though, that such a huge volume
of information has its limitations: the vast majority of genes currently being identified are of unknown function and their organism of origin is often a complete
mystery. Sometimes it is possible to find suitable phylogenetic markers associated
with DNA fragments, but more often than not, the precise origin of the genomic
sequence remains unknown. However, such challenges are now tractable, especially
with the advent of ultrasequencing approaches.
11
diversity has now been attempted with a variety of techniques including functional
and environmental genomics, transcriptomics (using microarrays and quantitative
PCR), proteomics and metabolic/metabolomic studies. Such studies have provided
crucial clues to the functional significance of natural biodiversity through the study
of well-known metabolic pathways.
Initially, perhaps the greatest success in marine functional genomics was
achieved using bacterial artificial chromosome (BAC) and fosmid (f1 origin-based
cosmid vector) libraries that allowed up to 300 kb of DNA inserts to be studied in
E. coli. A multiplex PCR screen for rRNA fragments allows the identification of
clones from specific phylogenetic groups. Full sequencing can then allow metabolic
genes to be identified in order to infer the metabolic potential of the specific organism or phylogenetic group. Such an advance was first achieved by Stein et al.
1996 with a marine Archaea population from coastal waters, followed by (Suzuki
et al. 2004)’s picoplankton study. BAC libraries derived from marine environments are now commonplace, yet still often require hundreds, if not thousands
of litres of seawater to produce. Recent modifications include the use of terminal restriction fragment length polymorphism (TRFLP) and internal transcribed
space, length heterogeneity PCR (ITS-LH-PCR) to screen for rRNA genes (Suzuki
et al. 2004, Babcock et al. 2007). Perhaps the greatest success story to come
from the BAC approach is the identification of a novel light driven photon pump
(now known as a bacteriorhodopsin) in an uncultured SAR86 (γ proteobacterial)
group BAC fragment (Beja et al. 2000). Since then the BAC approach has been
used with both natural marine communities and specific marine strains such as the
gammaproteobacterium Congregibacter litoralis (Fuchs et al. 2007), Eastern and
Pacific oysters Crassostrea virginica and C. gigas (Cunningham et al. 2006) and
protochordate Botryllus schlosseri (de Tomaso and Weissman 2003).
In addition to large-scale conventional sequencing approaches, the development
of pyrosequencing technologies now allows direct shotgun sequencing of environmental samples (Blow 2008) on a hitherto unforeseen scale (Huse et al. 2007,
Huber et al. 2007, Mou et al. 2008). Such metagenomic approaches (Handelsmann
2004) have had major implications for the marine environment with its massive
pool (quite literally) of unstudied and unculturable organisms. To date, combinations of chain-termination and pyrosequencing approaches have been used to study
marine bacterial, eukaryotic and viral diversity, sampled from diverse marine environments (coastal, open ocean, surface, deep sea, coral, subseafloor) (Breitbart et al.
2004, Venter et al. 2004, Angly et al. 2006, Culley et al. 2006, Sogin et al. 2006,
Bench et al. 2007, Biddle et al. 2008, Dinsdale et al. 2008, Quaiser et al. 2008,
Williamson et al. 2008). It is important to note though, that such a huge volume
of information has its limitations: the vast majority of genes currently being identified are of unknown function and their organism of origin is often a complete
mystery. Sometimes it is possible to find suitable phylogenetic markers associated
with DNA fragments, but more often than not, the precise origin of the genomic
sequence remains unknown. However, such challenges are now tractable, especially
with the advent of ultrasequencing approaches.