Marine Sponge Metagenomics 17.2 The Principle of Metagenomics and Related Techniques 463
Part C | 17.2
ins, siderophores, and anti-inflammatorials to immunosuppressants. These pharmacologically relevant bioactivities have motivated extensive searches for novel
NRPS genes in microbial isolates and in environmental samples. NRPS are large, multimodular enzymes
that are organized in modules containing specific domains that sequentially incorporate amino acid building blocks into a growing peptide chain. A typical
NRPS module contains an adenylation (A) domain,
a peptidyl carrier protein domain, and a condensation
domain [17.32].
17.2.5 Next Generation Sequencing
Technologies
Next generation sequencing (NGS) technologies are
having a considerable impact on metagenomics. NGS
technologies include three commercial platforms,
namely the 454 pyrosequencer (Roche Diagnostic
Corporation), GAII/HiSeq sequencer (Illumina), and
SOLiD instrument (Life Technologies Corporation).
The technologies behind each platform were reviewed
in detail by Metzker [17.34].
Although a huge amount of phylogenetic data has
been gathered for microbial identification using Sanger
sequencing, new sequencing technologies are having
a considerable impact on metagenomics. DNA pyrosequencing was developed to sequence the DNA
with a fundamentally different approach in the mid
1990s [17.35]. Sequencing occurs by a DNA polymerase driven production of inorganic pyrophosphate
with the formation of ATP and ATP-dependent conversion of luciferin to oxyluciferin (Fig. 17.3). The
oxyluciferin generate the emission of light pulses and
the amplitude of each signal is directly related to the
presence of one or more nucleosides. The main drawback of pyrosequencing is its inability to sequence
longer stretches of DNA. Hence, the hypervariable regions within bacterial 16S rRNA genes were amplified
by the PCR and subjected to DNA pyrosequencing.
DNA pyrosequencing has been effectively applied
in various fields such as genotyping, signle-nucleotide
polymorphism, and microbial identification [17.36].
Pyrosequencing has been effectively applied to identify microbes based on the hypervariable regions within
the 16S rRNA gene and signature sequence matching [17.37]. After DNA sequencing, sequences should
be analyzed carefully to facilitate accurate bacterial
identification.
Illumina performs solid surface PCR amplification
by immobilizing random DNA fragments on a surface.
The resulting cluster DNA fragments are sequenced
with reversible terminators by synthesis process. The
cluster density is enormous with hundreds of millions
sequence reads for each surface channel. In total, 16
channels are run at the same time in the HiSeq2000
instrument. The sequence read length is about 150 bp
and it can be sequenced from both ends. By overlapping both sequences, base pair reads can reach up to
300 bp. Approximately 60 Gbp can be expected in a single channel. As compared with 454, Illumina requires
only a few nanogram of starting material and provides
a superior amount of unassembled sequence reads. The
limitation of Illumina is the run time; it takes approximately 10 days but 454 take only 1 day [17.38].
Apart from this, some other sequencing technologies are also available, but they need to prove themselves for metagenomic application. Among them, the
Applied Biosystems SOLiD sequencer has been extensively used in genome re-sequencing. SOLiD possibly
provides the lowest error rate compared to other NGS
sequencing technologies, but the reliable read length is
much beyond 50 nucleotides. This will limit its applicability for direct gene annotation of unassembled reads
or assembly to large contigs. However, recent research
work on the porphyrins from a metagenomic library
of the marine sponge Discodermia calyx has showed
N encouraging outcome [17.39]. Ion Torrent is another emerging technology and is based on the principle
that nucleotide incorporation can be detected by proton release during DNA polymerization. This system
promises read lengths of more than 100 bp. Most recently, the draft genome sequence of Gillisia sp., strain
CBA3202 was announced based on the Ion Torrent Personal Genome Machine (PGM) sequencer [17.40].
17.2.6 Metagenomics and Informatics
The challenge for metagenomics is after getting hundreds or thousands of sequences, that is, how to
identify the microbes in reasonable time. Existing
bioinformatics methods are too slow and not adequate for large-scale projects. Currently, high-throughput metagenomics ribosomal DNA (rDNA) analysis
tools are in development. Obviously, enough computational command, distributed computing networks,
and robust server technology may ultimately solve the
demands of current metagenomics data analysis. Starting with sequence collection and verification, various
strategies have been followed to examine the quality of
each read [17.41]. Before sequence assembly, the sequences are trimmed by using various algorithms to
Part C | 17.2
ins, siderophores, and anti-inflammatorials to immunosuppressants. These pharmacologically relevant bioactivities have motivated extensive searches for novel
NRPS genes in microbial isolates and in environmental samples. NRPS are large, multimodular enzymes
that are organized in modules containing specific domains that sequentially incorporate amino acid building blocks into a growing peptide chain. A typical
NRPS module contains an adenylation (A) domain,
a peptidyl carrier protein domain, and a condensation
domain [17.32].
17.2.5 Next Generation Sequencing
Technologies
Next generation sequencing (NGS) technologies are
having a considerable impact on metagenomics. NGS
technologies include three commercial platforms,
namely the 454 pyrosequencer (Roche Diagnostic
Corporation), GAII/HiSeq sequencer (Illumina), and
SOLiD instrument (Life Technologies Corporation).
The technologies behind each platform were reviewed
in detail by Metzker [17.34].
Although a huge amount of phylogenetic data has
been gathered for microbial identification using Sanger
sequencing, new sequencing technologies are having
a considerable impact on metagenomics. DNA pyrosequencing was developed to sequence the DNA
with a fundamentally different approach in the mid
1990s [17.35]. Sequencing occurs by a DNA polymerase driven production of inorganic pyrophosphate
with the formation of ATP and ATP-dependent conversion of luciferin to oxyluciferin (Fig. 17.3). The
oxyluciferin generate the emission of light pulses and
the amplitude of each signal is directly related to the
presence of one or more nucleosides. The main drawback of pyrosequencing is its inability to sequence
longer stretches of DNA. Hence, the hypervariable regions within bacterial 16S rRNA genes were amplified
by the PCR and subjected to DNA pyrosequencing.
DNA pyrosequencing has been effectively applied
in various fields such as genotyping, signle-nucleotide
polymorphism, and microbial identification [17.36].
Pyrosequencing has been effectively applied to identify microbes based on the hypervariable regions within
the 16S rRNA gene and signature sequence matching [17.37]. After DNA sequencing, sequences should
be analyzed carefully to facilitate accurate bacterial
identification.
Illumina performs solid surface PCR amplification
by immobilizing random DNA fragments on a surface.
The resulting cluster DNA fragments are sequenced
with reversible terminators by synthesis process. The
cluster density is enormous with hundreds of millions
sequence reads for each surface channel. In total, 16
channels are run at the same time in the HiSeq2000
instrument. The sequence read length is about 150 bp
and it can be sequenced from both ends. By overlapping both sequences, base pair reads can reach up to
300 bp. Approximately 60 Gbp can be expected in a single channel. As compared with 454, Illumina requires
only a few nanogram of starting material and provides
a superior amount of unassembled sequence reads. The
limitation of Illumina is the run time; it takes approximately 10 days but 454 take only 1 day [17.38].
Apart from this, some other sequencing technologies are also available, but they need to prove themselves for metagenomic application. Among them, the
Applied Biosystems SOLiD sequencer has been extensively used in genome re-sequencing. SOLiD possibly
provides the lowest error rate compared to other NGS
sequencing technologies, but the reliable read length is
much beyond 50 nucleotides. This will limit its applicability for direct gene annotation of unassembled reads
or assembly to large contigs. However, recent research
work on the porphyrins from a metagenomic library
of the marine sponge Discodermia calyx has showed
N encouraging outcome [17.39]. Ion Torrent is another emerging technology and is based on the principle
that nucleotide incorporation can be detected by proton release during DNA polymerization. This system
promises read lengths of more than 100 bp. Most recently, the draft genome sequence of Gillisia sp., strain
CBA3202 was announced based on the Ion Torrent Personal Genome Machine (PGM) sequencer [17.40].
17.2.6 Metagenomics and Informatics
The challenge for metagenomics is after getting hundreds or thousands of sequences, that is, how to
identify the microbes in reasonable time. Existing
bioinformatics methods are too slow and not adequate for large-scale projects. Currently, high-throughput metagenomics ribosomal DNA (rDNA) analysis
tools are in development. Obviously, enough computational command, distributed computing networks,
and robust server technology may ultimately solve the
demands of current metagenomics data analysis. Starting with sequence collection and verification, various
strategies have been followed to examine the quality of
each read [17.41]. Before sequence assembly, the sequences are trimmed by using various algorithms to