36
A. Meyerdierks and F.O. Glöckner
et al. 1992), and bacterial artificial chromosomes (BACs) (Shizuya et al. 1992) (see
Section 2.3.5.2). The large inserts (> 30 kbp) in these clones offered the chance to
gain access to whole operons, and to find a phylogenetic marker on the same contiguous region (contig ) as a gene of interest, indicating the phylogenetic affiliation
of the microorganism from which the genomic fragment originated. The discovery
of proteorhodopsin based on large insert metagenomic libraries, by co-localisation
of a rhodopsin-like gene and a 16S rRNA gene affiliating with the γ-proteobacterial
SAR86 cluster on a 130 kb BAC clone, remains one of the most intriguing outcomes of these metagenomic studies (Beja et al. 2000, 2001). This and subsequent
studies changed substantially our understanding of aerobic, anoxygenic phototrophy in the oceans and its relevance to global carbon and energy budgets (Bryant
and Frigaard 2006). More recently, it has even been demonstrated that complete
bacterial genomes can be reconstructed, based on metagenomic fosmid clones. This
approach was used for a rice cluster I bacterium. For this project, about 3,700 fosmid
clones were constructed from an enrichment culture and used to assemble a complete genome (Erkel et al. 2006). Moreover, massive fosmid insert-end sequencing
has been applied to analyse depth-variable community trends in carbon and energy
metabolism of planktonic communities (DeLong et al. 2006).
With decreasing costs and improved high-throughput sequencing techniques,
metagenome analysis based on small insert libraries (about 1.5–3 kbp) became
“en vogue”. These approaches were particularly successful in low diversity habitats, for example in a study of the microbial community in an acid-mine drainage
(Tyson et al. 2004). In a second study, of the symbionts of the marine worm Olavius
algarvensis, small insert shotgun library sequencing was complemented with fosmid sequencing (Woyke et al. 2006). Both of these studies provided fascinating
insights into the genetic capabilities of the respective microbial communities and the
potential interactions of microbes with each other, and with their host. The largest
massive small insert metagenomic library sequencing experiment carried out to date
was recently completed by the Global Ocean Sampling expedition (Rusch et al.
2007, Yooseph et al. 2007). This study was a follow up to the Sargasso Sea shotgun
sequencing project (Venter et al. 2004), which had already led to the identification
of 148 new phylotypes, and 69,901 novel genes, including 782 new proteorhodopsin
genes.
Recently, a new DNA sequencing method, called pyrosequencing, has been
developed. The technique does not involve cloning and is therefore free of cloning
biases (Margulies et al. 2005). Third generation pyrosequencing technology generates sequences of just over 400 bp. This technique in its infancy with read length
of about 100 bp already proved to be valuable for the study of microbial diversity
(Leininger et al. 2006, Sogin et al. 2006) and metagenome (Edwards et al. 2006)
analyses.
In addition to inventions and improvements in the field of DNA sequencing,
isothermal multiple displacement amplification (MDA) using bacteriophage φ29polymerase has generated considerable interest from molecular ecologists. The
technique allows the amplification of genomic DNA from minute amounts of environmental samples to obtain insights into microbial communities such as those
A. Meyerdierks and F.O. Glöckner
et al. 1992), and bacterial artificial chromosomes (BACs) (Shizuya et al. 1992) (see
Section 2.3.5.2). The large inserts (> 30 kbp) in these clones offered the chance to
gain access to whole operons, and to find a phylogenetic marker on the same contiguous region (contig ) as a gene of interest, indicating the phylogenetic affiliation
of the microorganism from which the genomic fragment originated. The discovery
of proteorhodopsin based on large insert metagenomic libraries, by co-localisation
of a rhodopsin-like gene and a 16S rRNA gene affiliating with the γ-proteobacterial
SAR86 cluster on a 130 kb BAC clone, remains one of the most intriguing outcomes of these metagenomic studies (Beja et al. 2000, 2001). This and subsequent
studies changed substantially our understanding of aerobic, anoxygenic phototrophy in the oceans and its relevance to global carbon and energy budgets (Bryant
and Frigaard 2006). More recently, it has even been demonstrated that complete
bacterial genomes can be reconstructed, based on metagenomic fosmid clones. This
approach was used for a rice cluster I bacterium. For this project, about 3,700 fosmid
clones were constructed from an enrichment culture and used to assemble a complete genome (Erkel et al. 2006). Moreover, massive fosmid insert-end sequencing
has been applied to analyse depth-variable community trends in carbon and energy
metabolism of planktonic communities (DeLong et al. 2006).
With decreasing costs and improved high-throughput sequencing techniques,
metagenome analysis based on small insert libraries (about 1.5–3 kbp) became
“en vogue”. These approaches were particularly successful in low diversity habitats, for example in a study of the microbial community in an acid-mine drainage
(Tyson et al. 2004). In a second study, of the symbionts of the marine worm Olavius
algarvensis, small insert shotgun library sequencing was complemented with fosmid sequencing (Woyke et al. 2006). Both of these studies provided fascinating
insights into the genetic capabilities of the respective microbial communities and the
potential interactions of microbes with each other, and with their host. The largest
massive small insert metagenomic library sequencing experiment carried out to date
was recently completed by the Global Ocean Sampling expedition (Rusch et al.
2007, Yooseph et al. 2007). This study was a follow up to the Sargasso Sea shotgun
sequencing project (Venter et al. 2004), which had already led to the identification
of 148 new phylotypes, and 69,901 novel genes, including 782 new proteorhodopsin
genes.
Recently, a new DNA sequencing method, called pyrosequencing, has been
developed. The technique does not involve cloning and is therefore free of cloning
biases (Margulies et al. 2005). Third generation pyrosequencing technology generates sequences of just over 400 bp. This technique in its infancy with read length
of about 100 bp already proved to be valuable for the study of microbial diversity
(Leininger et al. 2006, Sogin et al. 2006) and metagenome (Edwards et al. 2006)
analyses.
In addition to inventions and improvements in the field of DNA sequencing,
isothermal multiple displacement amplification (MDA) using bacteriophage φ29polymerase has generated considerable interest from molecular ecologists. The
technique allows the amplification of genomic DNA from minute amounts of environmental samples to obtain insights into microbial communities such as those