Chapter 4
Metagenomic Analysis of Isotopically Enriched DNA
Yin Chen, Josh D. Neufeld, Marc G. Dumont, Michael W. Friedrich,
and J. Colin Murrell
Abstract
This detailed protocol describes an approach for combining DNA stable-isotope probing-based enrichment, multiple displacement amplification (MDA), and metagenomics. Together, these three methodologies enable selective access to the genomes of uncultivated organisms that actively grow using
isotopically labelled carbon and nitrogen sources. Incubations with stable-isotope-labelled substrates
enrich isotopically labelled DNA from functionally relevant micro-organisms; this serves as a filter to
reduce the complexity of the metagenome. The MDA step generates sufficient DNA from labelled nucleic
acid for metagenomic library construction. Subsequently, genome fragments can be subjected to a variety
of screens for phylogenetic or functional genes relevant to active community members. The MDAgenerated DNA can also serve as template for direct high-throughput sequencing to aid reconstruction
of metabolic pathways of those active organisms. Recent proof-of-concept studies have demonstrated
that this novel combination of molecular methods can offer substantial enhancements to gene detection
frequencies and may have great future potential for the discovery of novel genes, enzymes, and metabolic
pathways.
Key words: DNA stable-isotope probing, Multiple displacement amplification, Metagenomics
1. Introduction
Molecular ecology has revolutionised our understanding of
microbial diversity in the environment over the last two decades
as this approach has bypassed the limitations inherent to classical cultivation strategies. More than 600,000 sequences of the
ubiquitous marker SSU rRNA have been collected to date by
cultivation-independent surveys. And yet, the physiology and
functions of these uncultivated micro-organisms represented
only by 16S rRNA gene sequences are largely unknown. One
successful strategy to unravel the function of uncultivated
Wolfgang R. Streit and Rolf Daniel (eds.), Metagenomics: Methods and Protocols, Methods in Molecular Biology, vol. 668,
DOI 10.1007/978-1-60761-823-2_4, © Springer Science+Business Media, LLC 2010
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microbes is metagenomics, which represents the retrieval and
analysis of genome fragments from all community members in
an environmental sample (1). Metagenomic DNA containing
the 16S rRNA gene can reveal the phylogenetic affiliation of the
original micro-organism as well as adjacent enzyme-encoding
genes. These “functional” genes help deduce the potential role
of these micro-organisms in the environment. Conventional
metagenomic approaches involve cloning of extracted environmental DNA followed by sequence-based and/or functionbased screens. However, given the relative rarity of most
microbial community members, massive shotgun sequencing of
environmental DNA focuses on the most abundant species in a
given sample. For example, given an assumed underlying community structure for the Global Ocean Sampling expedition
dataset (2), only ~50% of the total community DNA has been
captured, despite substantial sequencing effort; nearly five times
the sequencing reads would be required to access 90% of the
diversity of these samples (3). Furthermore, function-based
screening for enzymes of relevance to industry, biotechnology,
and pharmaceutics may be challenging due to extremely lowtarget gene frequency. An alternative approach might be classical enrichment of individual populations, and thereby the
enrichment of genes of interest prior to metagenomic analysis.
Enrichment often results in the selection of micro-organisms
that are irrelevant to the natural environment but adapted best
to the enrichment conditions.
Stable-isotope probing of DNA (DNA-SIP) is a cultivationindependent method to selectively label micro-organisms that
can metabolise a specific stable-isotope-labelled substrate [e.g.
13
C, 15N; (4)]. Since its development, DNA-SIP has been widely
used to study micro-organisms involved in particular bioprocesses [reviewed in (5) and (6)]. When carried out under nearnatural conditions, SIP has the potential to select for labelled
genomes of active populations while minimising the extent of
enrichment bias (7). In combination with metagenomics, DNASIP facilitates the selective isolation of DNA from functionally
relevant micro-organisms to construct metagenomic libraries in a
directed fashion that has not been possible previously (5, 7–10).
This goal was first achieved by exposing a soil sample to 13CH4,
retrieving high-quality labelled DNA without damaging UV
exposure, and generating a modest BAC library for the discovery
of multiple clones with pmoA-containing operons (11). However,
the major concern of combining DNA-SIP with metagenomics
has been the challenge of obtaining sufficient 13C-labelled “heavy”
DNA for construction of a metagenomic library, without using
excessively high concentrations of substrate and reducing the risk
of enrichment bias. This has been recently overcome by applying
multiple displacement amplification [MDA; (12–14)] to gener-
Metagenomic Analysis of Isotopically Enriched DNA
69
Fig. 1. Overview of the combination of DNA-SIP, multiple displacement amplification, and metagenomics. Diagrams for
enzymatic treatment of MDA products were modified with permission from (23).
ate large quantities of DNA from minute quantities of 13C-labelled
“heavy” DNA (13, 14). Outlining this novel approach, we present here a detailed protocol for using MDA to prepare metagenomic libraries from DNA that has been “filtered” by SIP
pre-incubation (Fig. 1).
2. Materials
2.1. Reagents
and Equipments
for DNA-SIP
The reagents and equipment required for DNA-SIP have been
described elsewhere in detail, and we recommend readers to refer
to these protocols (15, 16). Briefly, reagents needed include:
1.Suitable stable-isotope (i.e. 13C or 15N) -labelled compounds.
2.CsCl stock solution (density 1.890 g/mL in water).
3.Gradient buffer: 0.1 M Tris–HCl, 0.1 M KCl, 1 mM EDTA,
pH 8.0.
4.DNA precipitation buffer: 30% polyethylene glycol 6000,
1.6 M NaCl.
5.Glycogen: 20 mg/mL in water.
6.TE buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 7.6.
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7.The key instruments for DNA-SIP include:
(a) Ultracentrifuge and corresponding rotor.
(b) Appropriate device for measuring substrate con­sumption.
(c) Suitable device for gradient fractionation.
(d) Digital refractometer (e.g. Reichert 2000™) or an analytic
balance for measuring gradient density.
2.2. Multiple
Displacement
Amplification
1.GenomiPhi V2 DNA Amplification Kit (GE Healthcare).
2.Thermal cycler.
3.500 mL PCR tubes.
4.Pipettors and corresponding tips.
5.Agarose.
2.3. Enzyme Treatment
of MDA-Generated
DNA
1.Phi29 DNA polymerase and corresponding buffer.
2.S1 nuclease and corresponding buffer.
3.DNA polymerase I and corresponding buffer.
4.Microcon YM-30 column (Millipore).
5.0.5 M Ethylene diamine tetraacetic acid (EDTA).
6.Phenol:chloroform:isoamyl alcohol (25:24:1 v/v; pH 8.0).
7.Chloroform:isoamyl alcohol (24:1 v/v).
8.Reagent-grade ethanol.
9.Microcentrifuge.
2.4. Metagenome
Library Construction
and Screening
1.CopyControl™ Fosmid Library Production Kit (Epicentre),
including End-Repair enzyme mix (Epicentre) and GELase
(Epicentre).
2.CHEF Mapper pulsed-field gel electrophoresis system (e.g.
Bio-Rad).
3.Low-melting point agarose.
3. Methods
3.1. DNA Extraction
and Preparation
of 13C-Labelled DNA
A proper DNA-SIP experiment includes the following key steps:
1.SIP incubation with stable-isotope compounds.
2.DNA extraction from the labelled environmental samples.
3.Ultracentrifugation and gradient fractionation.
4.DNA precipitation and identification of labelled “heavy”
DNA.
Metagenomic Analysis of Isotopically Enriched DNA
71
As mentioned in the previous section, it is not our intention
to present a detailed protocol for DNA-SIP set up in this chapter
and we refer readers to (15) and (16) to determine appropriate
incubation conditions and steps for purifying labelled DNA from
caesium chloride (CsCl) gradients. When 13C-labelled “heavy”
DNA is purified from CsCl gradient fractions, it serves as template for subsequent amplification and cloning within metagenomic libraries (see Note 1).
3.2. Multiple
Displacement
Amplification
In the initial study by Dumont et al. (11), a 13C-substrate concentration which exceeded that which was present in situ was chosen
to obtain the microgram quantities of DNA required for preparing
metagenomic libraries. The potential drawbacks of using elevated
substrate concentrations and long incubation times include an
enrichment bias and cross-feeding of substrate label to non-target
populations. Here, we describe an alternative protocol that uses
DNA retrieved from SIP incubations with near in situ concentrations of labelled substrate and short incubation times. The low
yields of labelled DNA from the DNA-SIP are then augmented
using MDA (Fig. 1).
1.1 mL of “heavy” DNA (13C-labelled), ideally ~1–10 ng, is
mixed with 9 mL of sample buffer (see Note 2).
2.The mixture is heated to 95°C for 3 min and then placed on
ice.
3.9 mL of reaction buffer and 1 mL of phi29 enzyme mix are
combined, and then added to the template and buffer mixture,
which is incubated at 30°C for 1.5 h for amplification.
4.The enzyme is inactivated by heating at 65°C for 10 min.
5.1 mL of the amplification product is loaded on a 1% (w/v)
agarose gel to quantify yield (see Note 3).
3.3. Enzyme Treatment
of MDA-Generated
DNA
MDA-generated DNA is hyperbranched and contains both doublestranded chimeras (which are stable) and chimeras that contain
single-stranded regions (which may be cleaved enzymatically); the
processes leading to chimera formation have been described in
(17). This study indicated that chimeras were formed by rearrangement of two neighbouring fragments (usually <10 kb in
distance in the genomic sequence), of which rearrangement of
inverted sequences with intervening deletions was the major
chimera type during MDA (~85%). In order to resolve the hyperbranched structure and eliminate single-stranded chimeras, the
reaction products may be treated enzymatically to generate doublestranded DNA suitable for cloning (Fig. 1). The enzyme treatment
process outlined below was initially introduced by Zhang et al.
(18) and is the most effective method tested for reducing the
occurrence of chimeras (see Note 4).
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Chen et al.
1.MDA-generated DNA is purified using a Microcon YM-30
column (Millipore) and washed with water to remove random
hexamer primers according to the manufacturer’s directions
(see Note 5).
2.The purified DNA (200 mL) is then incubated with 10 U of
phi29 DNA polymerase (Fermentas) at 30°C for 2 h without
adding random hexamer primers for debranching.
3.The reaction is stopped by heating at 65°C for 10 min.
4.The DNA is purified using another Microcon YM-30 column
(Millipore).
5.The purified DNA is eluted by 100 mL of water and then
incubated with 200 U of S1 nuclease (Fermentas) at 37°C for
30 min to digest single-stranded DNA.
6.The reaction is stopped by adding 50 mL of 0.5 M EDTA and
heating at 70°C for 10 min.
7.DNA is extracted with phenol:chloroform:isoamyl alcohol
(25:24:1, v/v), again with chloroform:isoamyl alcohol (24:1,
v/v), and then precipitated with ethanol (see Note 6).
8.The pellet is air-dried and dissolved in 100 mL of nucleasefree water, to which 20 U of DNA polymerase I and 0.4 mL
dNTP mix (25 mM each) are added for nick translation to
repair gaps within the DNA.
9.The reaction is incubated at 25°C for 1 h and then stopped
by heating at 75°C for 10 min.
3.4. Metagenome
Library Construction
and Screening
The enzyme-treated, MDA-generated DNA can be used for highthroughput sequencing (e.g. 454 pyrosequencing) or for construction of a metagenomic clone library (e.g. plasmid, cosmid/
fosmid, or BAC vectors). Readers are recommended to refer to
Mußmann et al. (19) for protocol details involving DNA preparation for direct high-throughput sequencing, which is not the focus
of this book chapter. Here, we describe a protocol for making a
fosmid library using the enzyme-treated, MDA-generated DNA.
1.After DNA polymerase I treatment and heat inactivation of
the enzyme, the DNA is end-repaired to generate blunt ends
using the End-Repair enzyme mix.
2.The end-repaired DNA is then loaded onto a 1% (w/v) lowmelting point agarose gel for size selection using a CHEF
Mapper pulsed-field gel electrophoresis system (see Notes 7
and 8).
3.Without UV exposure, a gel slice containing DNA fragments
of 30–50 kb is then excised from the agarose, and DNA is
purified from the gel using GELase and precipitated with
ethanol.
Metagenomic Analysis of Isotopically Enriched DNA
73
4.Fosmid library construction is carried out using the
CopyControl Fosmid Library Production Kit.
5.Routine sequence-based or function-based screening can be
carried out depending on the aim of the study (Fig. 1).
Sequence-based screening technologies are based on the known
gene sequences in public databases (e.g. GenBank) and therefore have limited success in the finding of novel genes. Functionbased approaches, on the other hand, are useful in the
identification of novel functions; however, they are extremely
limited by the fact that the genes from metagenomes must be
expressed, and the corresponding enzymes must be folded correctly in a heterologous background. Readers are recommended to refer to recent reviews and publications for updated
screening methods (1, 20, 21). Alternatively, the library can be
subjected to high-throughput sequencing to reconstruct
potential metabolic pathways of those micro-organisms that
metabolised the labelled substrate (see Note 9).
4. Notes
1.Methods for DNA extraction from DNA-SIP incubated
samples need to be selected carefully. For example, avoid
methods that shear DNA (e.g. bead-beating protocols) when
large-insert metagenomic libraries are desired. Purify the
DNA before loading into CsCl gradients if humic contaminants are present, even though CsCl gradient can partially
purify loaded DNA.
2.We found that phi29 DNA polymerase can be inhibited by
humic substances. We recommend that DNA extracted from
SIP incubations from soil samples be further purified (e.g. by
agarose gel purification) to remove any contaminants. This
will greatly enhance the yield of the amplification.
3.Typically, we found that at least 100 pg to 1 ng of DNA is
required as template for MDA using the GenomiPhi V2 kit.
However, lower starting template quantities (<1 ng) will yield
bias in the amplification process toward DNA from certain
organisms (13, 14, 18). Typically, ~4 mg of DNA will be generated from 1 ng of 13C-DNA in 2 h using this kit. Longer
incubations at 30°C or alternative MDA kits [reviewed in
(12)] will increase this yield if necessary.
4.To assess the potential bias of phi29 DNA polymerase during
MDA, we applied denaturing gradient gel electrophoresis
(DGGE) to compare fingerprints of 16S rRNA gene fragments before and after MDA (13, 14). Other methods such
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Chen et al.
as microarray hybridisation may also be used. This is highly
recommended since MDA is vulnerable to contamination
owing to its high sensitivity.
5.During the enzyme treatment process, ~50% of the DNA may
be lost. Thus, it is recommended that several MDA reactions
be combined before enzyme treatment if high yields are
critical. Alternatively, perform a second round of MDA using
1 mL of product from the first amplification as template. This
will greatly enhance the yield of the amplification process, but
potential bias introduced by this additional amplification step
should also be assessed (see Note 4).
6.A standard phenol:chloroform extraction and ethanol precipitation protocol can be used here (22). In addition, we
found that DNA recovery rate can be significantly increased
when a phase-lock tube (e.g. Eppendorf) is used.
7.Settings for pulsed-field gel electrophoresis using a CHEF
Mapper system (Bio-Rad) depend on the needed size of DNA
fragments. To select for fragments between 30 and 50 kb,
readers are recommended to refer to (14) and (13) for sample
run conditions.
8.Cloning efficiency can be significantly reduced if DNA is
exposed to UV light. If available, a Dark Reader transilluminator (Clare Chemical Research Inc.) should be used instead
of a standard UV transilluminator. Readers are also recommended to refer to the manual of CopyControl Fosmid
Library Production Kit (Epicentre) for a detailed protocol for
staining and size selection of sample DNA from low-melting
point agarose gel. Alternatively, DNA may be stained with
Sybr stain and the gel scanned using a fluorescence scanner,
such as that used for imaging 2D protein gels or DGGE
fingerprints (13, 14).
9.MDA-generated DNA will contain chimeras; this is not a
caveat that has been fully resolved. A recent study showed that
chimeras are produced during the amplification process itself
(17) and strategies to minimise chimera formation during
MDA need to be further improved.
Acknowledgements
Natural Environment Research Council (NERC), the Deutsche
Forschungsgemeinschaft (DFG), and the Max Planck Society
are acknowledged for financial support. JDN acknowledges
funding from a Discovery Grant from the National Sciences and
Engineering Research Council of Canada (NSERC).
Metagenomic Analysis of Isotopically Enriched DNA
75
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