libraries are significantly different. A first analysis will
determine if the bank is representative of the diversity
present in the sample. Then in a second step, the analysis compares two banks using the recovery value (coverage). The analysis can be done online (http://libshuff.
Mib.Uga.Edu/doc.Html). In general, researchers conduct
analysis whereas two sequences are different when they
have less than 97 % similarity, which is consistent with
the criteria for distinguishing the two species.
DNA Arrays
Sorting of sequences in a mixture of nucleic acids is most
often done by capillary electrophoresis which yields a pattern of presence/absence of data for all lengths of fragments.
An alternative is cloning/sequencing that is very expensive
and low speed but yields phylogenetic information for each
fragment analyzed. Before the development of
pyrosequencing, a compromise had emerged between the
two types of approaches with DNA microarrays that are
medium to high throughput, are relatively inexpensive, and
provide phylogenetic information and semiquantitative
abundance data. The chips are still not widely used: indeed,
whereas the per-use price is low, the necessary equipment is
expensive and available in only a few laboratories. For this
approach, the amplicons of a marker gene such as the 16S
rRNA gene are amplified by incorporating a fluorochrome
and hybridized on a glass slide with hundreds or thousands
of oligonucleotides designed after the study of known
sequences to represent a large number of individual bacterial
taxonomic groups. The detection is performed by laser excitation and fluorescence reading for each of the plots, and
quantification is made by comparison with a standardized
scale. However, these data should be viewed as semiquantitative. This method was used to compare rhizobacterial
community composition in soils suppressive or conducive
to tobacco black root rot disease (Kyselkova et al. 2009).
A Web of Science search with DNA arrays or microarrays as
keywords until December 2012 permitted to retrieve 95,838
references, though microbial ecology represents only a small
part of these papers. A strong increase in the use of this
method was observed during the 1998–2005 period, with a
maximum observed in 2010. However, a decrease in the use
of microarrays is observed since 2010 (Fig. 8.1).
Pyrosequencing: An Increasingly Used Method to
Quantify Microbial Diversity
The pyrosequencing technique is detailed in Sect. 18.1.2.
Shortly, it consists in amplifying a large number of targeted
sequences from genomic DNA and sequencing them, which
allows a direct access to sequence identity and relative abundance. This approach has been developed during the last
years, and it will greatly reduce the price of sequences and
make the acquisition of environmental sequences easier and
therefore more prevalent in microbial ecology projects. In
particular, the approach does not require any step of preselection of sequences by restriction analysis. Pyrosequencing
has been used to quantify microbial diversity in a range of
environments such as plant rhizospheres (Lundberg et al.
2012), the human intestinal tract (Tasse et al. 2010), or
marine biotopes (Bittner et al. 2013). A Web of Science
search with pyrosequencing as keyword until December
2012 permitted to retrieve 3,614 references. An exponential
increase in the use of this method was observed over the
2000–2012 period, and in 2012 this method became the
most widely used after microarrays (Fig. 8.1).
Approaches Targeting RNA
DNA is a stable molecule selected over evolution to be the
long-term memory of cell lineages. However, the stability of
the DNA molecule is such that DNA of inactive cells present
in a given biotope will be quantified as well as the DNA of
active cells. Moreover, the DNA of dead cells remains
detectable for years after cell death (Willerslev et al. 2004)
until lesions (alkylation, oxidation) accumulate, making
PCR less effective. However, there are now techniques that
are used to separate the DNA of viable cells from DNA that
is not associated with viable cells, prior to performing PCR
reactions (Nocker et al. 2007). If the aim is the characterization of active cells/communities in a given biotope, it is
possible to extract RNA from a given sample and study it.
However, RNA is a more unstable molecule that is recycled
rapidly by the cell so that the cell’s instructions are
modulated according to the changing environment. This
implies one needs to be particularly cautious when studying
environmental samples, for instance, by immediately freezing in liquid nitrogen the soil, sediment, or water samples in
the field just after harvesting from their natural habitats.
Functional Genes
In theory, any gene can be used to study, by the PCR-based
methods described above, the diversity of microorganisms
performing a function in a given environment. However,
various factors explain that some genes are more or less
interesting for this kind of approach. The first factor is the
sequence conservation required for primers to hybridize and
therefore amplify all genes associated with a given function
and only them. However, it is generally difficult to be certain
that all proteins performing a function are homologous: for
instance, the second step of denitrification (NO 2
À
) > NO)
is performed by two proteins, namely, the Cu-NIR enzyme
with a copper cofactor and the NIR-cd1 enzyme with a heme
cytochrome cofactor. These two proteins have no detectable
sequence similarity and the two should thus be targeted in
parallel (Coyne et al. 1989 ; Philippot et al. 2007). In other
8 Biodiversity and Microbial Ecosystems Functioning
273
determine if the bank is representative of the diversity
present in the sample. Then in a second step, the analysis compares two banks using the recovery value (coverage). The analysis can be done online (http://libshuff.
Mib.Uga.Edu/doc.Html). In general, researchers conduct
analysis whereas two sequences are different when they
have less than 97 % similarity, which is consistent with
the criteria for distinguishing the two species.
DNA Arrays
Sorting of sequences in a mixture of nucleic acids is most
often done by capillary electrophoresis which yields a pattern of presence/absence of data for all lengths of fragments.
An alternative is cloning/sequencing that is very expensive
and low speed but yields phylogenetic information for each
fragment analyzed. Before the development of
pyrosequencing, a compromise had emerged between the
two types of approaches with DNA microarrays that are
medium to high throughput, are relatively inexpensive, and
provide phylogenetic information and semiquantitative
abundance data. The chips are still not widely used: indeed,
whereas the per-use price is low, the necessary equipment is
expensive and available in only a few laboratories. For this
approach, the amplicons of a marker gene such as the 16S
rRNA gene are amplified by incorporating a fluorochrome
and hybridized on a glass slide with hundreds or thousands
of oligonucleotides designed after the study of known
sequences to represent a large number of individual bacterial
taxonomic groups. The detection is performed by laser excitation and fluorescence reading for each of the plots, and
quantification is made by comparison with a standardized
scale. However, these data should be viewed as semiquantitative. This method was used to compare rhizobacterial
community composition in soils suppressive or conducive
to tobacco black root rot disease (Kyselkova et al. 2009).
A Web of Science search with DNA arrays or microarrays as
keywords until December 2012 permitted to retrieve 95,838
references, though microbial ecology represents only a small
part of these papers. A strong increase in the use of this
method was observed during the 1998–2005 period, with a
maximum observed in 2010. However, a decrease in the use
of microarrays is observed since 2010 (Fig. 8.1).
Pyrosequencing: An Increasingly Used Method to
Quantify Microbial Diversity
The pyrosequencing technique is detailed in Sect. 18.1.2.
Shortly, it consists in amplifying a large number of targeted
sequences from genomic DNA and sequencing them, which
allows a direct access to sequence identity and relative abundance. This approach has been developed during the last
years, and it will greatly reduce the price of sequences and
make the acquisition of environmental sequences easier and
therefore more prevalent in microbial ecology projects. In
particular, the approach does not require any step of preselection of sequences by restriction analysis. Pyrosequencing
has been used to quantify microbial diversity in a range of
environments such as plant rhizospheres (Lundberg et al.
2012), the human intestinal tract (Tasse et al. 2010), or
marine biotopes (Bittner et al. 2013). A Web of Science
search with pyrosequencing as keyword until December
2012 permitted to retrieve 3,614 references. An exponential
increase in the use of this method was observed over the
2000–2012 period, and in 2012 this method became the
most widely used after microarrays (Fig. 8.1).
Approaches Targeting RNA
DNA is a stable molecule selected over evolution to be the
long-term memory of cell lineages. However, the stability of
the DNA molecule is such that DNA of inactive cells present
in a given biotope will be quantified as well as the DNA of
active cells. Moreover, the DNA of dead cells remains
detectable for years after cell death (Willerslev et al. 2004)
until lesions (alkylation, oxidation) accumulate, making
PCR less effective. However, there are now techniques that
are used to separate the DNA of viable cells from DNA that
is not associated with viable cells, prior to performing PCR
reactions (Nocker et al. 2007). If the aim is the characterization of active cells/communities in a given biotope, it is
possible to extract RNA from a given sample and study it.
However, RNA is a more unstable molecule that is recycled
rapidly by the cell so that the cell’s instructions are
modulated according to the changing environment. This
implies one needs to be particularly cautious when studying
environmental samples, for instance, by immediately freezing in liquid nitrogen the soil, sediment, or water samples in
the field just after harvesting from their natural habitats.
Functional Genes
In theory, any gene can be used to study, by the PCR-based
methods described above, the diversity of microorganisms
performing a function in a given environment. However,
various factors explain that some genes are more or less
interesting for this kind of approach. The first factor is the
sequence conservation required for primers to hybridize and
therefore amplify all genes associated with a given function
and only them. However, it is generally difficult to be certain
that all proteins performing a function are homologous: for
instance, the second step of denitrification (NO 2
À
) > NO)
is performed by two proteins, namely, the Cu-NIR enzyme
with a copper cofactor and the NIR-cd1 enzyme with a heme
cytochrome cofactor. These two proteins have no detectable
sequence similarity and the two should thus be targeted in
parallel (Coyne et al. 1989 ; Philippot et al. 2007). In other
8 Biodiversity and Microbial Ecosystems Functioning
273